Cytokine receptor common gamma chain like

ABSTRACT

The present invention relates to a novel human protein called Cytokine Receptor Common Gamma Chain Like, and isolated polynucleotides encoding this protein. Also provided are vectors, host cells, antibodies, and recombinant methods for producing this human protein. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to this novel human protein.

PRIORITY

This application is a continuation-in-part of U.S. application Ser. No.09/263,626, filed Mar. 5, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/078,563, filed Mar. 19,1998, and U.S. Provisional Application No. 60/086,505, filed May 22,1998, and is a continuation-in-part of International Application No.PCT/US99/05068, filed Mar. 5, 1999, all herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the Cytokine Receptor family. Morespecifically, the present invention relates to a polynucleotide encodinga novel human polypeptide named Cytokine Receptor Common Gamma ChainLike, or “CRCGCL.” This invention also relates to CRCGCL polypeptides,as well as vectors, host cells, antibodies directed to CRCGCLpolypeptides, and the recombinant methods for producing the same. Alsoprovided are diagnostic methods for detecting disorders related to theimmune system, and therapeutic methods for treating such disorders. Theinvention further relates to screening methods for identifying agonistsand antagonists of CRCGCL activity.

BACKGROUND OF THE INVENTION

Biochemical and physiological effects often result from the binding of acytokine to a specific receptor molecule. Receptor binding thenstimulates certain, and often independent, signal transduction pathways.(Kishimoto, T., et al., Cell 76:253-262 (1994.) The interaction betweena cytokine and a receptor is a primary regulator of a variety ofcellular processes, including activation, proliferation, anddifferentiation. (Arai, K.-I, et al., Ann. Rev. Biochem. 59:783-836(1990); Paul, W. E. and Seder, R. A., Cell 76:241-251 (1994)).

Cytokines that bind to the interleukin-2 (IL-2) receptor common gammachain (gamma c), including IL-2, IL-4, IL-7, IL-9, and IL-15, areimportant for the growth and differentiation of immune cells, such as Tand B lymphocytes, natural killer cells, macrophages, and monoctyes.These cytokines have overlapping biological effects that in part resultfrom the use of the shared receptor subunit gamma c. Recently it hasbeen shown that these cytokines activate a number of importantintracellular signaling molecules, by binding to the interleukin-2(IL-2) receptor common gamma chain (gamma c), including the Januskinases JAK1 and JAK3 and members of the transcription factor family ofsignal transducers and activators of transcription (STATs).

The discovery of these signaling pathways has led to important newinsights into their role in lymphocyte maturation, as it has emergedthat mutations in the genes encoding both gamma c and JAK3 result insimilar forms of severe combined immunodeficiency (SCID). For example,mutations in the human interleukin-2 (IL-2) receptor gamma, mapped tothe X chromosome, is associated with X-linked severe combinedimmunodeficiency. (Human Molecular Genetics, 2(8): 1099 (1993).)

Thus, there is a need for polypeptides that regulate the differentiationand proliferation of cells, since disturbances of such regulation may beinvolved in disorders relating to immune system. Therefore, there is aneed for identification and characterization of such human polypeptideswhich can play a role in detecting, preventing, ameliorating orcorrecting such disorders.

SUMMARY OF THE INVENTION

The present invention relates to a novel polynucleotide and the encodedpolypeptide of CRCGCL. Moreover, the present invention relates tovectors, host cells, antibodies, and recombinant methods for producingthe polypeptides and polynucleotides. Also provided are diagnosticmethods for detecting disorders relates to the polypeptides, andtherapeutic methods for treating such disorders. The invention furtherrelates to screening methods for identifying binding partners of CRCGCL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the nucleotide sequence (SEQ ID NO:1) and the deducedamino acid sequence (SEQ ID NO:2) of CRCGCL. The predicted leadersequence is located at about amino acids 1-22.

FIG. 2 shows the regions of identity between the amino acid sequence ofthe CRCGCL protein and the translation product of the closest homolog,the Bos Taurus Interleukin-2 receptor gamma (Accession Nos. 1532088)(SEQ ID NO:3), determined by BLAST analysis. Identical amino acidsbetween the two polypeptides are shaded in black, while conservativeamino acids are boxed. By examining the regions of amino acids shadedand/or boxed, the skilled artisan can readily identify conserved domainsbetween the two polypeptides. These conserved domains are preferredembodiments of the present invention.

FIG. 3 shows an analysis of the CRCGCL amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown, and all were generated using the default settings. In the“Antigenic Index or Jameson-Wolf” graph, the positive peaks indicatelocations of the highly antigenic regions of the CRCGCL protein, i.e.,regions from which epitope-bearing peptides of the invention can beobtained. The domains defined by these graphs are contemplated by thepresent invention. Tabular representation of the data summarizedgraphically in FIG. 3 can be found in Table 1. The columns are labeledwith the headings “Res”, “Position”, and Roman Numerals I-XIV. Thecolumn headings refer to the following features of the amino acidsequence presented in FIG. 3, and Table 1: “Res”: amino acid residue ofSEQ ID NO:2 and FIGS. 1A and 1B; “Position”: position of thecorresponding residue within SEQ ID NO:2 and FIGS. 1A and 1B; I: Alpha,Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta,Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn,Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Gamier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

DETAILED DESCRIPTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.

In the present invention, a “secreted” CRCGCL protein refers to aprotein capable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as aCRCGCL protein released into the extracellular space without necessarilycontaining a signal sequence. If the CRCGCL secreted protein is releasedinto the extracellular space, the CRCGCL secreted protein can undergoextracellular processing to produce a “mature” CRCGCL protein. Releaseinto the extracellular space can occur by many mechanisms, includingexocytosis and proteolytic cleavage.

As used herein, a CRCGCL “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:1 or the cDNA containedwithin the clone deposited with the ATCC. For example, the CRCGCLpolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a CRCGCL“polypeptide” refers to a molecule having the translated amino acidsequence generated from the polynucleotide as broadly defined. However,one embodiment of the present invention does not include thepolynucleotide sequence of Genbank Accession No. X91553, hereinincorporated by reference.

In specific embodiments, the polynucleotides of the invention are lessthan 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.In a further embodiment, polynucleotides of the invention comprise atleast 15 contiguous nucleotides of CRCGCL coding sequence, but do notcomprise all or a portion of any CRCGCL intron. In another embodiment,the nucleic acid comprising CRCGCL coding sequence does not containcoding sequences of a genomic flanking gene (i.e., 5′ or 3′ to theCRCGCL gene in the genome).

In the present invention, the full length CRCGCL sequence identified asSEQ ID NO:1 was generated by overlapping sequences of the depositedclone (contig analysis). A representative clone containing all or mostof the sequence for SEQ ID NO:1 was deposited with the American TypeCulture Collection (“ATCC”) on Mar. 23, 1998, and was given the ATCCDeposit Number 209691. A second clone was also deposited with the ATCCon Feb. 25, 1998, and given ATCC Deposit Number 209641. The ATCC islocated at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.The ATCC deposit was made pursuant to the terms of the Budapest Treatyon the international recognition of the deposit of microorganisms forpurposes of patent procedure.

A CRCGCL “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO:1, the complement thereof, or the cDNA within thedeposited clone. “Stringent hybridization conditions” refers to anovernight incubation at 42 degree C. in a solution comprising 50%formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to theCRCGCL polynucleotides at moderatetly high stringency hybridizationconditions. Changes in the stringency of hybridization and signaldetection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example,moderately high stringency conditions include an overnight incubation at37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2MNaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmonsperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1%SDS. In addition, to achieve even lower stringency, washes performedfollowing stringent hybridization can be done at higher saltconcentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

The CRCGCL polynucleotide can be composed of any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, CRCGCL polynucleotides can be composed ofsingle- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the CRCGCL polynucleotides can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. CRCGCLpolynucleotides may also contain one or more modified bases or DNA orRNA backbones modified for stability or for other reasons. “Modified”bases include, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

CRCGCL polypeptides can be composed of amino acids joined to each otherby peptide bonds or modified peptide bonds, i.e., peptide isosteres, andmay contain amino acids other than the 20 gene-encoded amino acids. TheCRCGCL polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theCRCGCL polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in a given CRCGCL polypeptide. Also, a givenCRCGCL polypeptide may contain many types of modifications. CRCGCLpolypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic CRCGCL polypeptides may resultfrom posttranslation natural processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a CRCGCL polynucleotide sequence while “SEQ IDNO:2” refers to a CRCGCL polypeptide sequence.

A CRCGCL polypeptide “having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a CRCGCL polypeptide, including mature forms, as measured ina particular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof the CRCGCL polypeptide, but rather substantially similar to thedose-dependence in a given activity as compared to the CRCGCLpolypeptide (i.e., the candidate polypeptide will exhibit greateractivity or not more than about 25-fold less and, preferably, not morethan about tenfold less activity, and most preferably, not more thanabout three-fold less activity relative to the CRCGCL polypeptide.)

CRCGCL Polynucleotides and Polypeptides

Clone HTAEK53 was isolated from an activated T-cell cDNA library.Initially, the sequence of clone HTAEK53 was identified as SEQ ID NO:26and the deduced amino acid sequence was predicted as SEQ ID NO:27, witha recognition that an apparent frame shift in the sequence existed. Thisframe shift was easily resolved using standard molecular biologytechniques, generating the nucleotide sequence of SEQ ID NO:1 and thededuced amino acid sequence shown in SEQ ID NO:2.

The deposited clone contains a cDNA having a total of 1573 nucleotides,which encodes a predicted open reading frame of 371 amino acid residues.(See FIGS. 1A-1B.) The open reading frame begins at a N-terminalmethionine located at nucleotide position 13, and ends at a stop codonat nucleotide position 1128. The predicted molecular weight of theCRCGCL protein should be about 42 kDa.

Subsequent Northern analysis also showed a 1.6 Kb transcript in acervical cancer cell line (HeLa), activated T cells, and a lungcarcinoma cell line (A549), while a shorter variant is also expressed inthe lymph node and to a lesser extent in the spleen tissues, a patternconsistent with immune specific expression.

CRCGCL expression was not observed in the following cell lines, HL60,K562, Molt-4, Raji, SW480, G361, as well as the heart, brain, placenta,lung, liver, skeletal muscle, kidney, pancreas, thymus, prostate,testis, ovary, small intestine, colon, or peripheral blood leukocytes, apattern consistent with immune specific expression.

Using BLAST analysis, SEQ ID NO:2 was found to be homologous to membersof the Cytokine Receptor family. Particularly, SEQ ID NO:2 containsdomains homologous to the translation product of the Bos Taurus mRNA forInterleukin-2 receptor gamma (Accession Nos. 1532088) (FIG. 2) (SEQ IDNO:3), including the following conserved domains: (a) a predictedtransmembrane domain domain located at about amino acids 226-260; (b) apredicted WXWS (SEQ ID NO:30), or [STGL]-x-W-[SG]-x-W-S (SEQ ID NO:18),domain located at about amino acids 198-204 (T-x-P-S-x-W-S) (SEQ IDNO:19), although not a perfect match; and (c) a predicted Jak Box,having the motif W(P,E)X(V,I)P(N,S,D)P (SEQ ID NO:20), domain located atabout amino acids 261-268 (I-P-X-V-P-D-P) (SEQ ID NO:21), although not aperfect match. These polypeptide fragments of CRCGCL are specificallycontemplated in the present invention. Because Interleukin-2 receptorgamma (Accession Nos. 1532088) is thought to be important as a cytokinereceptor, the homology between Interleukin-2 receptor gamma (AccessionNos. 1532088) and CRCGCL suggests that CRCGCL may also be involved inthe differentiation and proliferation of cells. CRCGCL is alsohomologous to other Interleukin-2 receptor gamma genes isolated from avariety of species, such as human (Accession No. gi/349632), Canisfamiliaris (Accession No. gi/517412), and mouse (pri/S37582).

Moreover, the encoded polypeptide has a predicted leader sequencelocated at about amino acids 1-22. (See FIGS. 1A-1B.) Also shown inFIGS. 1A-1B, one embodiment of the secreted form of CRCGCL encompassesabout amino acids 23-371, amino acids 23-225, or amino acids 1-231.These polypeptide fragments of CRCGCL are specifically contemplated inthe present invention.

Other preferred polypeptide fragments comprise the amino acids sequence:QIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTS GC (SEQ ID NO:22);RRHSLFLHQEWDAPRFHRKSLDGLLPETQF (SEQ ID NO:23);LLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWVRVKAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPK (SEQ ID NO:24); and/orMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKPKLSKFILISSLAILLMVSLLLLSLWKLWRXKKFLXPSVPDPKSIFPGLFXIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAESPRMLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVA (SEQ ID NO:25), as well as fragments thereof.Also preferred are polynucleotide fragments encoding these polypeptidefragments.

Because CRCGCL was isolated from activated T cells, nucleic acids of theinvention are useful as reagents for differential identification of thetissue(s) or cell type(s) present in a biological sample and fordiagnosis of immune disorders. Similarly, polypeptides and antibodiesdirected to those polypeptides are useful to provide immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the immune system, expression of this geneat significantly higher or lower levels may be detected in certaintissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,serum, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue from anindividual not having the disorder.

The tissue distribution in only activated T-cells and homology to thecytokine receptors IL2 and IL13 suggests that this protein is a novelmember of the cytokine receptor family expressed specifically onT-cells. The tissue distribution of this gene in cells of the immunesystem suggests that the protein product of this clone would be usefulfor treatment, prophylaxis and diagnosis of immune and autoimmunediseases, such as lupus, transplant rejection, allergic reactions,arthritis, asthma, immunodeficiency diseases, leukemia, AIDS. Inaddition its expression in T-cells suggests a potential role in thetreatment, prophylaxis and detection of thymus disorders such as GravesDisease, lymphocytic thyroiditis, hyperthyroidism and hypothyroidism.The receptor could also serve as a target for small molecule ormonoclonal antibody, blocking its activity, which could be important inthe disease states listed herein.

The CRCGCL nucleotide sequence identified as SEQ ID NO:1 was assembledfrom partially homologous (“overlapping”) sequences obtained from thedeposited clone, and in some cases, from additional related DNA clones.The overlapping sequences were assembled into a single contiguoussequence of high redundancy (usually three to five overlapping sequencesat each nucleotide position), resulting in a final sequence identifiedas SEQ ID NO:1.

Therefore, SEQ ID NO:1 and the translated SEQ ID NO:2 are sufficientlyaccurate and otherwise suitable for a variety of uses well known in theart and described further below. For instance, SEQ ID NO:1 is useful fordesigning nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO:1 or the cDNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 may be used to generate antibodies whichbind specifically to CRCGCL.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2, but also a sample of plasmid DNA containing a human cDNA ofCRCGCL deposited with the ATCC. The nucleotide sequence of the depositedCRCGCL clone can readily be determined by sequencing the deposited clonein accordance with known methods. The predicted CRCGCL amino acidsequence can then be verified from such deposits. Moreover, the aminoacid sequence of the protein encoded by the deposited clone can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell containing the deposited human CRCGCL cDNA,collecting the protein, and determining its sequence.

The present invention also relates to the CRCGCL gene corresponding toSEQ ID NO:1, SEQ ID NO:2, or the deposited clone. The CRCGCL gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the CRCGCL genefrom appropriate sources of genomic material.

Also provided in the present invention are species homologs of CRCGCL.Species homologs may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source for the desired homologue.

The CRCGCL polypeptides can be prepared in any suitable manner. Suchpolypeptides include isolated naturally occurring polypeptides,recombinantly produced polypeptides, synthetically producedpolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides are well understood inthe art.

The CRCGCL polypeptides may be in the form of the secreted protein,including the mature form, or may be a part of a larger protein, such asa fusion protein (see below). It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

CRCGCL polypeptides are preferably provided in an isolated form, andpreferably are substantially purified. A recombinantly produced versionof a CRCGCL polypeptide, including the secreted polypeptide, can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). CRCGCL polypeptides also can be purifiedfrom natural or recombinant sources using antibodies of the inventionraised against the CRCGCL protein in methods which are well known in theart.

Polynucleotide and Polypeptide Variants

“Variant” refers to a polynucleotide or polypeptide differing from theCRCGCL polynucleotide or polypeptide, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the CRCGCL polynucleotide or polypeptide.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the CRCGCLpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of SEQ ID NO:1, the ORF (open reading frame),or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the present invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.(1990) 6:237-245.) In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequences shown in SEQ ID NO:2 or to the amino acid sequenceencoded by deposited DNA clone can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).In a sequence alignment the query and subject sequences are either bothnucleotide sequences or both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The CRCGCL variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.CRCGCL polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli).

Naturally occurring CRCGCL variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level. Alternatively,non-naturally occurring variants may be produced by mutagenesistechniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the CRCGCL polypeptides. For instance, one or moreamino acids can be deleted from the N-terminus or C-terminus of thesecreted protein without substantial loss of biological function. Theauthors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reportedvariant KGF proteins having heparin binding activity even after deleting3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferongamma exhibited up to ten times higher activity after deleting 8-10amino acid residues from the carboxy terminus of this protein. (Dobeliet al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes CRCGCL polypeptide variants whichshow substantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, (e.g., encoding a polypeptide having the amino acidsequence of an N and/or C terminal deletion disclosed below as m-n ofSEQ ID NO:2), irrespective of whether they encode a polypeptide havingCRCGCL functional activity. This is because even where a particularnucleic acid molecule does not encode a polypeptide having CRCGCLfunctional activity, one of skill in the art would still know how to usethe nucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving CRCGCL functional activity include, inter alia, (1) isolating aCRCGCL gene or allelic or splice variants thereof in a cDNA library; (2)in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the CRCGCL gene, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988); and (3) Northern Blot analysis for detectingCRCGCL mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, which do, in fact, encode a polypeptide having CRCGCLfunctional activity. By “a polypeptide having CRCGCL functionalactivity” is intended polypeptides exhibiting activity similar, but notnecessarily identical, to a functional activity of the CRCGCLpolypeptides of the present invention (e.g., complete (full-length)CRCGCL, mature CRCGCL and soluble CRCGCL (e.g., having sequencescontained in the extracellular domain of CRCGCL) as measured, forexample, in a particular immunoassay or biological assay. For example, aCRCGCL functional activity can routinely be measured by determining theability of a CRCGCL polypeptide to bind a CRCGCL ligand. CRCGCLfunctional activity may also be measured by determining the ability of apolypeptide, such as cognate ligand which is free or expressed on a cellsurface, to induce cells expressing the polypeptide.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the depositedcDNA, the nucleic acid sequence shown in FIGS. 1A-1B (SEQ ID NO:1), orfragments thereof, will encode polypeptides “having CRCGCL functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having CRCGCLfunctional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

For example, site directed changes at the amino acid level of CRCGCL canbe made by replacing a particular amino acid with a conservative aminoacid. Preferred conservative mutations include: M1 replaced with A, G,I, L, S, T, or V; G2 replaced with A, I, L, S, T, M, or V; R3 replacedwith H, or K; L4 replaced with A, G, I, S, T, M, or V; V5 replaced withA, G, I, L, S, T, or M; L6 replaced with A, G, I, S, T, M, or V; L7replaced with A, G, I, S, T, M, or V; W8 replaced with F, or Y; G9replaced with A, I, L, S, T, M, or V; A10 replaced with G, I, L, S, T,M, or V; A11 replaced with G, I, L, S, T, M, or V; V12 replaced with A,G, I, L, S, T, or M; F13 replaced with W, or Y; L14 replaced with A, G,I, S, T, M, or V; L15 replaced with A, G, I, S, T, M, or V; G16 replacedwith A, I, L, S, T, M, or V; G17 replaced with A, I, L, S, T, M, or V;W18 replaced with F, or Y; M19 replaced with A, G, I, L, S, T, or V; A20replaced with G, I, L, S, T, M, or V; L21 replaced with A, G, I, S, T,M, or V; G22 replaced with A, I, L, S, T, M, or V; Q23 replaced with N;G24 replaced with A, I, L, S, T, M, or V; G25 replaced with A, I, L, S,T, M, or V; A26 replaced with G, I, L, S, T, M, or V; A27 replaced withG, I, L, S, T, M, or V; E28 replaced with D; G29 replaced with A, I, L,S, T, M, or V; V30 replaced with A, G, I, L, S, T, or M; Q31 replacedwith N; 132 replaced with A, G, L, S, T, M, or V; Q33 replaced with N;I34 replaced with A, G, L, S, T, M, or V; I35 replaced with A, G, L, S,T, M, or V; Y36 replaced with F, or W; F37 replaced with W, or Y; N38replaced with Q; L39 replaced with A, G, I, S, T, M, or V; E40 replacedwith D; T41 replaced with A, G, I, L, S, M, or V; V42 replaced with A,G, I, L, S, T, or M; Q43 replaced with N; V44 replaced with A, G, I, L,S, T, or M; T45 replaced with A, G, I, L, S, M, or V; W46 replaced withF, or Y; N47 replaced with Q; A48 replaced with G; I, L, S, T, M, or V;S49 replaced with A, G, I, L, T, M, or V; K50 replaced with H, or R; Y51replaced with F, or W; S52 replaced with A, G, I, L, T, M, or V; R53replaced with H, or K; T54 replaced with A, G, I, L, S, M, or V; N55replaced with Q; L56 replaced with A, G, I, S, T, M, or V; T57 replacedwith A, G, I, L, S, M, or V; F58 replaced with W, or Y; H59 replacedwith K, or R; Y60 replaced with F, or W; R61 replaced with H, or K; F62replaced with W, or Y; N63 replaced with Q; G64 replaced with A, I, L,S, T, M, or V; D65 replaced with E; E66 replaced with D; A67 replacedwith G, I, L, S, T, M, or V; Y68 replaced with F, or W; D69 replacedwith E; Q70 replaced with N; T72 replaced with A, G, I, L, S, M, or V;N73 replaced with Q; Y74 replaced with F, or W; L75 replaced with A, G,I, S, T, M, or V; L76 replaced with A, G, I, S, T, M, or V; Q77 replacedwith N; E78 replaced with D; G79 replaced with A, I, L, S, T, M, or V;H80 replaced with K, or R; T81 replaced with A, G, I, L, S, M, or V; S82replaced with A, G, I, L, T, M, or V; G83 replaced with A, I, L, S, T,M, or V; L85 replaced with A, G, I, S, T, M, or V; L86 replaced with A,G, I, S, T, M, or V; D87 replaced with E; A88 replaced with G, I, L, S,T, M, or V; E89 replaced with D; Q90 replaced with N; R91 replaced withH, or K; D92 replaced with E; D93 replaced with E; I94 replaced with A,G, L, S, T, M, or V; L95 replaced with A, G, I, S, T, M, or V; Y96replaced with F, or W; F97 replaced with W, or Y; S98 replaced with A,G, I, L, T, M, or V; I99 replaced with A, G, L, S, T, M, or V; R100replaced with H, or K; N101 replaced with Q; G102 replaced with A, I, L,S, T, M, or V; T103 replaced with A, G, I, L, S, M, or V; H104 replacedwith K, or R; V106 replaced with A, G, I, L, S, T, or M; F107 replacedwith W, or Y; T108 replaced with A, G, I, L, S, M, or V; A109 replacedwith G, I, L, S, T, M, or V; S110 replaced with A, G, I, L, T, M, or V;R111 replaced with H, or K; W112 replaced with F, or Y; M113 replacedwith A, G, I, L, S, T, or V; V114 replaced with A, G, I, L, S, T, or M;Y115 replaced with F, or W; Y116 replaced with F, or W; L117 replacedwith A, G, I, S, T, M, or V; K118 replaced with H, or R; S120 replacedwith A, G, I, L, T, M, or V; S121 replaced with A, G, I, L, T, M, or V;K123 replaced with H, or R; H124 replaced with K, or R; V125 replacedwith A, G, I, L, S, T, or M; R126 replaced with H, or K; F127 replacedwith W, or Y; S128 replaced with A, G, I, L, T, M, or V; W129 replacedwith F, or Y; H130 replaced with K, or R; Q131 replaced with N; D132replaced with E; A133 replaced with G, I, L, S, T, M, or V; V134replaced with A, G, I, L, S, T, or M; T135 replaced with A, G, I, L, S,M, or V; V136 replaced with A, G, I, L, S, T, or M; T137 replaced withA, G, I, L, S, M, or V; S139 replaced with A, G, I, L, T, M, or V; D140replaced with E; L141 replaced with A, G, I, S, T, M, or V; S142replaced with A, G, I, L, T, M, or V; Y143 replaced with F, or W; G144replaced with A, I, L, S, T, M, or V; D145 replaced with E; L146replaced with A, G, I, S, T, M, or V; L147 replaced with A, G, I, S, T,M, or V; Y148 replaced with F, or W; E149 replaced with D; V150 replacedwith A, G, I, L, S, T, or M; Q151 replaced with N; Y152 replaced with F,or W; R153 replaced with H, or K; S154 replaced with A, G, I, L, T, M,or V; F156 replaced with W, or Y; D157 replaced with E; T158 replacedwith A, G, I, L, S, M, or V; E159 replaced with D; W160 replaced with F,or Y; Q161 replaced with N; S162 replaced with A, G, I, L, T, M, or V;K163 replaced with H, or R; Q164 replaced with N; E165 replaced with D;N166 replaced with Q; T167 replaced with A, G, I, L, S, M, or V; N169replaced with Q; V170 replaced with A, G, I, L, S, T, or M; T171replaced with A, G, I, L, S, M, or V; I172 replaced with A, G, L, S, T,M, or V; E173 replaced with D; G174 replaced with A, I, L, S, T, M, orV; L175 replaced with A, G, I, S, T, M, or V; D176 replaced with E; A177replaced with G, I, L, S, T, M, or V; E178 replaced with D; K179replaced with H, or R; Y181 replaced with F, or W; S182 replaced with A,G, I, L, T, M, or V; F183 replaced with W, or Y; W184 replaced with F,or Y; V185 replaced with A, G, I, L, S, T, or M; R186 replaced with H,or K; V187 replaced with A, G, I, L, S, T, or M; K188 replaced with H,or R; A189 replaced with G, I, L, S, T, M, or V; M190 replaced with A,G, I, L, S, T, or V; E191 replaced with D; D192 replaced with E; V193replaced with A, G, I, L, S, T, or M; Y194 replaced with F, or W; G195replaced with A, I, L, S, T, M, or V; D197 replaced with E; T198replaced with A, G, I, L, S, M, or V; Y199 replaced with F, or W; S201replaced with A, G, I, L, T, M, or V; D202 replaced with E; W203replaced with F, or Y; S204 replaced with A, G, I, L, T, M, or V; E205replaced with D; V206 replaced with A, G, I, L, S, T, or M; T207replaced with A, G, I, L, S, M, or V; W209 replaced with F, or Y; Q210replaced with N; R211 replaced with H, or K; G212 replaced with A, I, L,S, T, M, or V; E213 replaced with D; I214 replaced with A, G, L, S, T,M, or V; R215 replaced with H, or K; D216 replaced with E; A217 replacedwith G, I, L, S, T, M, or V; A219 replaced with G, I, L, S, T, M, or V;E220 replaced with D; T221 replaced with A, G, I, L, S, M, or V; T223replaced with A, G, I, L, S, M, or V; K226 replaced with H, or R; K228replaced with H, or R; L229 replaced with A, G, I, S, T, M, or V; S230replaced with A, G, I, L, T, M, or V; K231 replaced with H, or R; F232replaced with W, or Y; I233 replaced with A, G, L, S, T, M, or V; L234replaced with A, G, I, S, T, M, or V; I235 replaced with A, G, L, S, T,M, or V; S236 replaced with A, G, I, L, T, M, or V; S237 replaced withA, G, I, L, T, M, or V; L238 replaced with A, G, I, S, T, M, or V; A239replaced with G, I, L, S, T, M, or V; I240 replaced with A, G, L, S, T,M, or V; L241 replaced with A, G, I, S, T, M, or V; L242 replaced withA, G, I, S, T, M, or V; M243 replaced with A, G, I, L, S, T, or V; V244replaced with A, G, I, L, S, T, or M; S245 replaced with A, G, I, L, T,M, or V; L246 replaced with A, G, I, S, T, M, or V; L247 replaced withA, G, I, S, T, M, or V; L248 replaced with A, G, I, S, T, M, or V; L249replaced with A, G, I, S, T, M, or V; S250 replaced with A, G, I, L, T,M, or V; L251 replaced with A, G, I, S, T, M, or V; W252 replaced withF, or Y; K253 replaced with H, or R; L254 replaced with A, G, I, S, T,M, or V; W255 replaced with F, or Y; R256 replaced with H, or K; V257replaced with A, G, I, L, S, T, or M; K258 replaced with H, or R; K259replaced with H, or R; F260 replaced with W, or Y; L261 replaced with A,G, I, S, T, M, or V; I262 replaced with A, G, L, S, T, M, or V; S264replaced with A, G, I, L, T, M, or V; V265 replaced with A, G, I, L, S,T, or M; D267 replaced with E; K269 replaced with H, or R; S270 replacedwith A, G, I, L, T, M, or V; I271 replaced with A, G, L, S, T, M, or V;F272 replaced with W, or Y; G274 replaced with A, I, L, S, T, M, or V;L275 replaced with A, G, I, S, T, M, or V; F276 replaced with W, or Y;E277 replaced with D; I278 replaced with A, G, L, S, T, M, or V; H279replaced with K, or R; Q280 replaced with N; G281 replaced with A, I, L,S, T, M, or V; N282 replaced with Q; F283 replaced with W, or Y; Q284replaced with N; E285 replaced with D; W286 replaced with F, or Y; I287replaced with A, G, L, S, T, M, or V; T288 replaced with A, G, I, L, S,M, or V; D289 replaced with E; T290 replaced with A, G, I, L, S, M, orV; Q291 replaced with N; N292 replaced with Q; V293 replaced with A, G,I, L, S, T, or M; A294 replaced with G, I, L, S, T, M, or V; H295replaced with K, or R; L296 replaced with A, G, I, S, T, M, or V; H297replaced with K, or R; K298 replaced with H, or R; M299 replaced with A,G, I, L, S, T, or V; A300 replaced with G, I, L, S, T, M, or V; G301replaced with A, I, L, S, T, M, or V; A302 replaced with G, I, L, S, T,M, or V; E303 replaced with D; Q304 replaced with N; E305 replaced withD; S306 replaced with A, G, I, L, T, M, or V; G307 replaced with A, I,L, S, T, M, or V; E309 replaced with D; E310 replaced with D; L312replaced with A, G, I, S, T, M, or V; V313 replaced with A, G, I, L, S,T, or M; V314 replaced with A, G, I, L, S, T, or M; Q315 replaced withN; L316 replaced with A, G, I, S, T, M, or V; A317 replaced with G, I,L, S, T, M, or V; K318 replaced with H, or R; T319 replaced with A, G,I, L, S, M, or V; E320 replaced with D; A321 replaced with G, I, L, S,T, M, or V; E322 replaced with D; S323 replaced with A, G, I, L, T, M,or V; R325 replaced with H, or K; M326 replaced with A, G, I, L, S, T,or V; L327 replaced with A, G, I, S, T, M, or V; D328 replaced with E;Q330 replaced with N; T331 replaced with A, G, I, L, S, M, or V; E332replaced with D; E333 replaced with D; K334 replaced with H, or R; E335replaced with D; A336 replaced with G, I, L, S, T, M, or V; S337replaced with A, G, I, L, T, M, or V; G338 replaced with A, I, L, S, T,M, or V; G339 replaced with A, I, L, S, T, M, or V; S340 replaced withA, G, I, L, T, M, or V; L341 replaced with A, G, I, S, T, M, or V; Q342replaced with N; L343 replaced with A, G, I, S, T, M, or V; H345replaced with K, or R; Q346 replaced with N; L348 replaced with A, G, I,S, T, M, or V; Q349 replaced with N; G350 replaced with A, I, L, S, T,M, or V; G351 replaced with A, I, L, S, T, M, or V; D352 replaced withE; V353 replaced with A, G, I, L, S, T, or M; V354 replaced with A, G,I, L, S, T, or M; T355 replaced with A, G, I, L, S, M, or V; I356replaced with A, G, L, S, T, M, or V; G357 replaced with A, I, L, S, T,M, or V; G358 replaced with A, I, L, S, T, M, or V; F359 replaced withW, or Y; T360 replaced with A, G, I, L, S, M, or V; F361 replaced withW, or Y; V362 replaced with A, G, I, L, S, T, or M; M363 replaced withA, G, I, L, S, T, or V; N364 replaced with Q; D365 replaced with E; R366replaced with H, or K; S367 replaced with A, G, I, L, T, M, or V; Y368replaced with F, or W; V369 replaced with A, G, I, L, S, T, or M; A370replaced with G, I, L, S, T, M, or V; L371 replaced with A, G, I, S, T,M, or V.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased CRCGCL activityor function, while the remaining CRCGCL activities or functions aremaintained. More preferably, the resulting constructs have more than oneincreased CRCGCL activity or function, while the remaining CRCGCLactivities or functions are maintained.

Besides conservative amino acid substitution, variants of CRCGCL include(i) substitutions with one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, CRCGCL polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

For example, preferred non-conservative substitutions of CRCGCL include:M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G2 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; R3 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; L4 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V5 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L7replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W8 replaced with D,E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G9 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; A10 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; A11 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; V12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F13replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L14replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L15 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G16 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G17 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; W18 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; M19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A20replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L21 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G22 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q23 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; G24 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; G25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A26replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A27 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; E28 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G29 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; V30 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q31 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; I32 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Q33 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; I34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I35 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y36 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F37 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N38 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L39 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; E40 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; T41 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; V42 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Q43 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; V44 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T45replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W46 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N47 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A48 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S49 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; K50 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Y51 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; S52 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R53 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N55 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T57 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F58 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H59 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y60 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R61 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F62 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N63 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G64 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; D65 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; E66 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; A67 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Y68 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; D69 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; Q70 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; C71 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; T72 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; N73 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; Y74 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; L75 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q77replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E78 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; G79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H80 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T81 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S82 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; G83 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; L85 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L86 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D87replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q90replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;R91 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;D92 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; D93 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; I94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L95replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y96 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F97 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S98 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I99 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R100 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; N101 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; G102 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; T103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;H104 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;P105 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; V106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F107replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T108replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A109 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S110 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R111 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; W112 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; M113 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y115replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y116replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;L117replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K118 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P119 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S120replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S121 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P122 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K123 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H124 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V125 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R126 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; F127 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; S128 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; W129 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; H130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; Q131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; D132 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; A133 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; V134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T135replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V136 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T137 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C138 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; S139 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; D140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; L141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y143replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G144replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D145 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L146 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L147 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Y148 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; E149 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; V150 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; Q151 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; Y152 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; R153 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; S154 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; P155 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; F156 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; D157 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E159 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; W160 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; Q161 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; S162 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K163replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q164replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E165 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N166 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; T167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C168replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; N169 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; V170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T171replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I172 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; E173 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G174 replaced with D, E, H, K,R, N, Q, F, W, Y, P, o r C; L175 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; D176 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; A177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; E178 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; K179 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; C180 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or P; Y181 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; S182 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F183 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;W184 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;V185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R186 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V187 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; K188 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A189 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; M190 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E191 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; D192 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; V193 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Y194 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; G195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P196replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; D197 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; T198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y199replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P200replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; S201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D202replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;W203 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;S204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E205 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V206replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T207 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C208 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; W209 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q210 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R211 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G212 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; E213 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; I214 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; R215 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; D216 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; A217 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; C218 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or P; A219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E220 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; T221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P222replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; T223 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P224replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; P225 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; K226 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; P227 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; K228 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; L229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K231 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F232 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I233 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L234 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I235 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S237 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L238 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A239 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I240 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M243 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V244 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S245 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L246 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L247 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L248 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L249 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S250 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;W252 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;K253 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L254 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W255 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R256 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V257 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; K258 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K259 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F260 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L261 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I262 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P263 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; S264 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; V265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P266replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; D267 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; P268 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; K269 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; S270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F272 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P273 replacedwith D, E, H, K, R, A, G,1, L, S, T, M, V, N, Q, F, W, Y, or C; G274replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L275 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F276 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; E277 replaced with H, K, R, A,G,1, L, S, T, M, V, N, Q, F, W, Y, P, or C; I278 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; H279 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q280 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; G281 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; N282 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; F283 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; Q284 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; E285 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; W286 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; I287 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; T288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D289 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; T290 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q291replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;N292 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; V293 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A294replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H295 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L296 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H297 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; K298 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; M299 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; A300 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G301 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A302replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E303 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q304 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E305replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S306 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G307 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P308 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E309 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E310 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P311 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L312replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V313 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V314 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q315 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; L316 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A317 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;K318 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;T319 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E320 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A321replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E322 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S323 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P324 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R325 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M326 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; L327 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; D328 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; P329 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; Q330 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; T331 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; E332 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; E333 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; K334 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; E335 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; A336 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; S337 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G338 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G339 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S340 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L341 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Q342 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; L343 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; P344 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; H345 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; Q346 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; P347 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, or C; L348 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Q349 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; G350 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G351replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D352 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V353 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V354 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T355 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; I356 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G357 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G358 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F359 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T360 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F361 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; V362 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M363 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N364 replaced with D, E, H, K, R, A, G, 1, L, S, T, M, V, F, W, Y, P, orC; D365 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; R366 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; S367 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y368replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V369replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A370 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L371 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have loss of a CRCGCL activity orfunction, while the remaining CRCGCL activities or functions aremaintained. More preferably, the resulting constructs have more than oneloss of CRCGCL activity or function, while the remaining CRCGCLactivities or functions are maintained.

Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and10) can be replaced with the substituted amino acids as described above(either conservative or nonconservative). The substituted amino acidscan occur in the full length, mature, or proprotein form of CRCGCLprotein, as well as the N- and C-terminal deletion mutants, having thegeneral formula m-n, listed below.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a CRCGCL polypeptide having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of a CRCGCL polypeptide, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of FIGS. 1A-1B or fragments thereof(e.g., the mature form and/or other fragments described herein), is 1-5,5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutionsare preferable.

Polynucleotide and Polypeptide Fragments

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having, for example, the nucleotide sequence ofthe deposited cDNA (clone HTAEK53), a nucleotide sequence encoding thepolypeptide sequence encoded by the deposited cDNA, a nucleotidesequence encoding the polypeptide sequence depicted in FIGS. 1A-1B (SEQID NO:2), the nucleotide sequence shown in FIGS. 1A-1B (SEQ ID NO:1), orthe complementary strand thereto, is intended fragments at least 15 nt,and more preferably at least about 20 nt, still more preferably at least30 nt, and even more preferably, at least about 40, 50, 100, 150, 200,250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length. Thesefragments have numerous uses that include, but are not limited to,diagnostic probes and primers as discussed herein. Of course, largerfragments, such as those of 501-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNA(clone HTAEK53) or as shown in FIGS. 1A-1B (SEQ ID NO:1). By a fragmentat least 20 nt in length, for example, is intended fragments whichinclude 20 or more contiguous bases from, for example, the nucleotidesequence of the deposited cDNA, or the nucleotide sequence as shown inFIGS. 1A-1B (SEQ ID NO:1).

Moreover, representative examples of CRCGCL polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350,351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800,800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450,1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, and/or 2001 tothe end of SEQ ID NO:1 or the complementary strand thereto, or the cDNAcontained in the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a CRCGCL functional activity. By apolypeptide demonstrating a CRCGCL “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) CRCGCL protein. Suchfunctional activities include, but are not limited to, biologicalactivity, antigenicity [ability to bind (or compete with a CRCGCLpolypeptide for binding) to an anti-CRCGCL antibody], immunogenicity(ability to generate antibody which binds to a CRCGCL polypeptide),ability to form multimers with CRCGCL polypeptides of the invention, andability to bind to a receptor or ligand for a CRCGCL polypeptide.

The functional activity of CRCGCL polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length CRCGCL polypeptide for binding toanti-CRCGCL antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a CRCGCL ligand is identified, or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol.Rev. 59:94-123. In another embodiment, physiological correlates ofCRCGCL binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability of CRCGCLpolypeptides and fragments, variants derivatives and analogs thereof toelicit CRCGCL related biological activity (either in vitro or in vivo).Other methods will be known to the skilled artisan and are within thescope of the invention.

The present invention is further directed to fragments of the CRCGCLpolypeptide described herein. By a fragment of an isolated the CRCGCLpolypeptide, for example, encoded by the deposited cDNA (clone HTAEK53),the polypeptide sequence encoded by the deposited cDNA, the polypeptidesequence depicted in FIGS. 1A-1B (SEQ ID NO:2), is intended to encompasspolypeptide fragments contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be at least 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind CRCGCL ligand) maystill be retained. For example, the ability of shortened CRCGCL muteinsto induce and/or bind to antibodies which recognize the complete ormature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan CRCGCL mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six CRCGCL amino acid residues may oftenevoke an immune response.

Accordingly, polypeptide fragments include the secreted CRCGCL proteinas well as the mature form. Further preferred polypeptide fragmentsinclude the secreted CRCGCL protein or the mature form having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of either the secretedCRCGCL polypeptide or the mature form. Similarly, any number of aminoacids, ranging from 1-30, can be deleted from the carboxy terminus ofthe secreted CRCGCL protein or mature form. Furthermore, any combinationof the above amino and carboxy terminus deletions are preferred.Similarly, polynucleotide fragments encoding these CRCGCL polypeptidefragments are also preferred.

Particularly, N-terminal deletions of the CRCGCL polypeptide can bedescribed by the general formula m-371, where m is an integer from 2 to370, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues of N-terminaldeletions of the CRCGCL polypeptide of the invention shown as SEQ IDNO:2, including polypeptides comprising the amino acid sequence ofresidues: G-2 to L-371; R-3 to L-371; L-4 to L-371; V-5 to L-371; L-6 toL-371; L-7 to L-371; W-8 to L-371; G-9 to L-371; A-10 to L-371; A-11 toL-371; V-12 to L-371; F-13 to L-371; L-14 to L-371; L-15 to L-371; G-16to L-371; G-17 to L-371; W-18 to L-371; M-19 to L-371; A-20 to L-371;L-21 to L-371; G-22 to L-371; Q-23 to L-371; G-24 to L-371; G-25 toL-371; A-26 to L-371; A-27 to L-371; E-28 to L-371; G-29 to L-371; V-30to L-371; Q-31 to L-371; I-32 to L-371; Q-33 to L-371; I-34 to L-371;I-35 to L-371; Y-36 to L-371; F-37 to L-371; N-38 to L-371; L-39 toL-371; E-40 to L-371; T-41 to L-371; V-42 to L-371; Q-43 to L-371; V-44to L-371; T-45 to L-371; W-46 to L-371; N-47 to L-371; A-48 to L-371;S-49 to L-371; K-50 to L-371; Y-51 to L-371; S-52 to L-371; R-53 toL-371; T-54 to L-371; N-55 to L-371; L-56 to L-371; T-57 to L-371; F-58to L-371; H-59 to L-371; Y-60 to L-371; R-61 to L-371; F-62 to L-371;N-63 to L-371; G-64 to L-371; D-65 to L-371; E-66 to L-371; A-67 toL-371; Y-68 to L-371; D-69 to L-371; Q-70 to L-371; C-71 to L-371; T-72to L-371; N-73 to L-371; Y-74 to L-371; L-75 to L-371; L-76 to L-371;Q-77 to L-371; E-78 to L-371; G-79 to L-371; H-80 to L-371; T-81 toL-371; S-82 to L-371; G-83 to L-371; C-84 to L-371; L-85 to L-371; L-86to L-371; D-87 to L-371; A-88 to L-371; E-89 to L-371; Q-90 to L-371;R-91 to L-371; D-92 to L-371; D-93 to L-371; I-94 to L-371; L-95 toL-371; Y-96 to L-371; F-97 to L-371; S-98 to L-371; I-99 to L-371; R-100to L-371; N-101 to L-371; G-102 to L-371; T-103 to L-371; H-104 toL-371; P-105 to L-371; V-106 to L-371; F-107 to L-371; T-108 to L-371;A-109 to L-371; S-110 to L-371; R-111 to 371; W-112 to L-371; M-113 toL-371; V-114 to L-371; Y-115 to L-371; Y-116 to L-371; L-117 to L-371;K-118 to L-371; P-119 to L-371; S-120 to L-371; S-121 to L-371; P-122 toL-371; K-123 to L-371; H-124 to L-371; V-125 to L-371; R-126 to L-371;F-127 to L-371; S-128 to L-371; W-129 to L-371; H-130 to L-371; Q-131 toL-371; D-132 to L-371; A-133 to L-371; V-134 to L-371; T-135 to L-371;V-136 to L-371; T-137 to L-371; C-138 to L-371; S-139 to L-371; D-140 toL-371; L-141 to L-371; S-142 to L-371; Y-143 to L-371; G-144 to L-371;D-145 to L-371; L-146 to L-371; L-147 to L-371; Y-148 to L-371; E-149 toL-371; V-150 to L-371; Q-151 to L-371; Y-152 to L-371; R-153 to L-371;S-154 to L-371; P-155 to L-371; F-156 to L-371; D-157 to L-371; T-158 toL-371; E-159 to L-371; W-160 to L-371; Q-161 to L-371; S-162 to L-371;K-163 to L-371; Q-164 to L-371; E-165 to L-371; N-166 to L-371; T-167 toL-371; C-168 to L-371; N-169 to L-371; V-170 to L-371; T-171 to L-371;I-172 to L-371; E-173 to L-371; G-174 to L-371; L-175 to L-371; D-176 toL-371; A-177 to L-371; E-178 to L-371; K-179 to L-371; C-180 to L-371;Y-181 to L-371; S-182 to L-371; F-183 to L-371; W-184 to L-371; V-185 toL-371; R186 to L-371; V-187 to L-371; K-188 to L-371; A-189 to L-371;M-190 to L-371; E-191 to L-371; D-192 to L-371; V-193 to L-371; Y-194 toL-371; G-195 to L-371; P-196 to L-371; D-197 to L-371; T-198 to L-371;Y-199 to L-371; P-200 to L-371; S-201 to L-371; D-202 to L-371; W-203 toL-371; S-204 to L-371; E-205 to L-371; V-206 to L-371; T-207 to L-371;C-208 to L-371; W-209 to L-371; Q-210 to L-371; R-211 to L-371; G-212 toL-371; E-213 to L-371; I-214 to L-371; R-215 to L-371; D-216 to L-371;A-217 to L-371; C-218 to L-371; A-219 to L-371; E-220 to L-371; T-221 toL-371; P-222 to L-371; T-223 to L-371; P-224 to L-371; P-225 to L-371;K-226 to L-371; P-227 to L-371; K-228 to L-371; L-229 to L-371; S-230 toL-371; K-231 to L-371; F-232 to L-371; I-233 to L-371; L-234 to L-371;I-235 to L-371; S-236 to L-371; S-237 to L-371; L-238 to L-371; A-239 toL-371; I-240 to L-371; L-241 to L-371; L-242 to L-371; M-243 to L-371;V-244 to L-371; S-245 to L-371; L-246 to L-371; L-247 to L-371; L-248 toL-371; L-249 to L-371; S-250 to L-371; L-251 to L-371; W-252 to L-371;K-253 to L-371; L-254 to L-371; W-255 to L-371; R-256 to L-371; V-257 toL-371; K-258 to L-371; K-259 to L-371; F-260 to L-371; L-261 to L-371;I-262 to L-371; P-263 to L-371; S-264 to L-371; V-265 to L-371; P-266 toL-371; D-267 to L-371; P-268 to L-371; K-269 to L-371; S-270 to L-371;I-271 to L-371; F-272 to L-371; P-273 to L-371; G-274 to L-371; L-275 toL-371; F-276 to L-371; E-277 to L-371; I-278 to L-371; H-279 to L-371;Q-280 to L-371; G-281 to L-371; N-282 to L-371; F-283 to L-371; Q-284 toL-371; E-285 to L-371; W-286 to L-371; I-287 to L-371; T-288 to L-371;D-289 to L-371; T-290 to L-371; Q-291 to L-371; N-292 to L-371; V-293 toL-371; A-294 to L-371; H-295 to L-371; L-296 to L-371; H-297 to L-371;K-298 to L-371; M-299 to L-371; A-300 to L-371; G-301 to L-371; A-302 toL-371; E-303 to L-371; Q-304 to L-371; E-305 to L-371; S-306 to L-371;G-307 to L-371; P-308 to L-371; E-309 to L-371; E-310 to L-371; P-311 toL-371; L-312 to L-371; V-313 to L-371; V-314 to L-371; Q-315 to L-371;L-316 to L-371; A-317 to L-371; K-318 to L-371; T-319 to L-371; E-320 toL-371; A-321 to L-371; E-322 to L-371; S-323 to L-371; P-324 to L-371;R-325 to L-371; M-326 to L-371; L-327 to L-371; D-328 to L-371; P-329 toL-371; Q-330 to L-371; T-331 to L-371; E-332 to L-371; E-333 to L-371;K-334 to L-371; E-335 to L-371; A-336 to L-371; S-337 to L-371; G-338 toL-371; G-339 to L-371; S-340 to L-371; L-341 to L-371; Q-342 to L-371;L-343 to L-371; P-344 to L-371; H-345 to L-371; Q-346 to L-371; P-347 toL-371; L-348 to L-371; Q-349 to L-371; G-350 to L-371; G-351 to L-371;D-352 to L-371; V-353 to L-371; V-354 to L-371; T-355 to L-371; I-356 toL-371; G-357 to L-371; G-358 to L-371; F-359 to L-371; T-360 to L-371;F-361 to L-371; V-362 to L-371; M-363 to L-371; N-364 to L-371; D-365 toL-371; R-366 to L-371; of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g, biological activities, ability to multimerize, ability to bindCRCGCL ligand) may still be retained. For example the ability of theshortened CRCGCL mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an CRCGCL mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixCRCGCL amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the CRCGCL polypeptide shown in FIGS. 1A-1B (SEQ ID NO:2),as described by the general formula 1-n, where n is an integer from 2 to371, where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues of C-terminaldeletions of the CRCGCL polypeptide of the invention shown as SEQ IDNO:2 include polypeptides comprising the amino acid sequence ofresidues: M-1 to A-370; M-1 to V-369; M-1 to Y-368; M-1 to S-367; M-1 toR-366; M-1 to D-365; M-1 to N-364; M-1 to M-363; M-1 to V-362; M-1 toF-361; M-1 to T-360; M-1 to F-359; M-1 to G-358; M-1 to G-357; M-1 toI-356; M-1 to T-355; M-1 to V-354; M-1 to V-353; M-1 to D-352; M-1 toG-351; M-1 to G-350; M-1 to Q-349; M-1 to L-348; M-1 to P-347; M-1 toQ-346; M-1 to H-345; M-1 to P-344; M-1 to L-343; M-1 to Q-342; M-1 toL-341; M-1 to S-340; M-1 to G-339; M-1 to G-338; M-1 to S-337; M-1 toA-336; M-1 to E-335; M-1 to K-334; M-1 to E-333; M-1 to E-332; M-1 toT-331; M-1 to Q-330; M-1 to P-329; M-1 to D-328; M-1 to L-327; M-1 toM-326; M-1 to R-325; M-1 to P-324; M-1 to S-323; M-1 to E-322; M-1 toA-321; M-1 to E-320; M-1 to T-319; M-1 to K-318; M-1 to A-317; M-1 toL-316; M-1 to Q-315; M-1 to V-314; M-1 to V-313; M-1 to L-312; M-1 toP-311; M-1 to E-310; M-1 to E-309; M-1 to P-308; M-1 to G-307; M-1 toS-306; M-1 to E-305; M-1 to Q-304; M-1 to E-303; M-1 to A-302; M-1 toG-301; M-1 to A-300; M-1 to M-299; M-1 to K-298; M-1 to H-297; M-1 toL-296; M-1 to H-295; M-1 to A-294; M-1 to V-293; M-1 to N-292; M-1 toQ-291; M-1 to T-290; M-1 to D-289; M-1 to T-288; M-1 to I-287; M-1 toW-286; M-1 to E-285; M-1 to Q-284; M-1 to F-283; M-1 to N-282; M-1 toG-281; M-1 to Q-280; M-1 to H-279; M-1 to I-278; M-1 to E-277; M-1 toF-276; M-1 to L-275; M-1 to G-274; M-1 to P-273; M-1 to F-272; M-1 toI-271; M-1 to S-270; M-1 to K-269; M-1 to P-268; M-1 to D-267; M-1 toP-266; M-1 to V-265; M-1 to S-264; M-1 to P-263; M-1 to I-262; M-1 toL-261; M-1 to F-260; M-1 to K-259; M-1 to K-258; M-1 to V-257; M-1 toR-256; M-1 to W-255; M-1 to L-254; M-1 to K-253; M-1 to W-252; M-1 toL-251; M-1 to S-250; M-1 to L-249; M-1 to L-248; M-1 to L-247; M-1 toL-246; M-1 to S-245; M-1 to V-244; M-1 to M-243; M-1 to L-242; M-1 toL-241; M-1 to I-240; M-1 to A-239; M-1 to L-238; M-1 to S-237; M-1 toS-236; M-1 to I-235; M-1 to L-234; M-1 to I-233; M-1 to F-232; M-1 toK-231; M-1 to S-230; M-1 to L-229; M-1 to K-228; M-1 to P-227; M-1 toK-226; M-1 to P-225; M-1 to P-224; M-1 to T-223; M-1 to P-222; M-1 toT-221; M-1 to E-220; M-1 to A-219; M-1 to C-218; M-1 to A-217; M-1 toD-216; M-1 to R-215; M-1 to I-214; M-1 to E-213; M-1 to G-212; M-1 toR-211; M-1 to Q-210; M-1 to W-209; M-1 to C-208; M-1 to T-207; M-1 toV-206; M-1 to E-205; M-1 to S-204; M-1 to W-203; M-1 to D-202; M-1 toS-201; M-1 to P-200; M-1 to Y-199; M-1 to T-198; M-1 to D-197; M-1 toP-196; M-1 to G-195; M-1 to Y-194; M-1 to V-193; M-1 to D-192; M-1 toE-191; M-1 to M-190; M-1 to A-189; M-1 to K-188; M-1 to V-187; M-1 toR-186; M-1 to V-185; M-1 to W-184; M-1 to F-183; M-1 to S-182; M-1 toY-181; M-1 to C-180; M-1 to K-179; M-1 to E-178; M-1 to A-177; M-1 toD-176; M-1 to L-175; M-1 to G-174; M-1 to E-173; M-1 to I-172; M-1 toT-171; M-1 to V-170; M-1 to N-169; M-1 to C-168; M-1 to T-167; M-1 toN-166; M-1 to E-165; M-1 to Q-164; M-1 to K-163; M-1 to S-162; M-1 toQ-161; M-1 to W-160; M-1 to E-159; M-1 to T-158; M-1 to D-157; M-1 toF-156; M-1 to P-155; M-1 to S-154; M-1 to R-153; M-1 to Y-152; M-1 toQ-151; M-1 to V-150; M-1 to E-149; M-1 to Y-148; M-1 to L-147; M-1 toL-146; M-1 to D-145; M-1 to G-144; M-1 to Y-143; M-1 to S-142; M-1 toL-141; M-1 to D-140; M-1 to S-139; M-1 to C-138; M-1 to T-137; M-1 toV-136; M-1 to T-135; M-1 to V-134; M-1 to A-133; M-1 to D-132; M-1 toQ-131; M-1 to H-130; M-1 to W-129; M-1 to S-128; M-1 to F-127; M-1 toR-126; M-1 to V-125; M-1 to H-124; M-1 to K-123; M-1 to P-122; M-1 toS-121; M-1 to S-120; M-1 to P-119; M-1 to K-118; M-1 to L-117; M-1 toY-116; M-1 to Y-115; M-1 to V-114; M-1 to M-113; M-1 to W-112; M-1 toR-111; M-1 to S-110; M-1 to A-109; M-1 to T-108; M-1 to F-107; M-1 toV-106; M-1 to P-105; M-1 to H-104; M-1 to T-103; M-1 to G-102; M-1 toN-101; M-1 to R-100; M-1 to I-99; M-1 to S-98; M-1 to F-97; M-1 to Y-96;M-1 to L-95; M-1 to I-94; M-1 to D-93; M-1 to D-92; M-1 to R-91; M-1 toQ-90; M-1 to E-89; M-1 to A-88; M-1 to D-87; M-1 to L-86; M-1 to L-85;M-1 to C-84; M-1 to G-83; M-1 to S-82; M-1 to T-81; M-1 to H-80; M-1 toG-79; M-1 to E-78; M-1 to Q-77; M-1 to L-76; M-1 to L-75; M-1 to Y-74;M-1 to N-73; M-1 to T-72; M-1 to C-71; M-1 to Q-70; M-1 to D-69; M-1 toY-68; M-1 to A-67; M-1 to E-66; M-1 to D-65; M-1 to G-64; M-1 to N-63;M-1 to F-62; M-1 to R-61; M-1 to Y-60; M-1 to H-59; M-1 to F-58; M-1 toT-57; M-1 to L-56; M-1 to N-55; M-1 to T-54; M-1 to R-53; M-1 to S-52;M-1 to Y-51; M-1 to K-50; M-1 to S-49; M-1 to A-48; M-1 to N-47; M-1 toW-46; M-1 to T-45; M-1 to V-44; M-1 to Q-43; M-1 to V-42; M-1 to T-41;M-1 to E-40; M-1 to L-39; M-1 to N-38; M-1 to F-37; M-1 to Y-36; M-1 toI-35; M-1 to I-34; M-1 to Q-33; M-1 to I-32; M-1 to Q-31; M-1 to V-30;M-1 to G-29; M-1 to E-28; M-1 to A-27; M-1 to A-26; M-1 to G-25; M-1 toG-24; M-1 to Q-23; M-1 to G-22; M-1 to L-21; M-1 to A-20; M-1 to M-19;M-1 to W-18; M-1 to G-17; M-1 to G-16; M-1 to L-15; M-1 to L-14; M-1 toF-13; M-1 to V-12; M-1 to A-11; M-1 to A-10; M-1 to G-9; M-1 to W-8; M-1to L-7; of SEQ ID NO:2. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can becombined to produce a N- and C-terminal deleted CRCGCL polypeptide. Theinvention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues m-n of SEQ ID NO:2, where n and mare integers as described above. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

For example, N- and C-terminal deletion mutants comprising the solubledomain of SEQ ID NO:2 are also contemplated. In particular, theinvention provides polynucleotides encoding polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues ofN-terminal deletions of the CRCGCL polypeptide of the invention shown asSEQ ID NO:2, including polypeptides comprising the amino acid sequenceof residues: G-2 to K-231; R-3 to K-231; L-4 to K-231; V-5 to K-231; L-6to K-231; L-7 to K-231; W-8 to K-231; G-9 to K-231; A-10 to K-231; A-11to K-231; V-12 to K-231; F-13 to K-231; L-14 to K-231; L-15 to K-231;G-16 to K-231; G-17 to K-231; W-18 to K-231; M-19 to K-231; A-20 toK-231; L-21 to K-231; G-22 to K-231; Q-23 to K-231; G-24 to K-231; G-25to K-231; A-26 to K-231; A-27 to K-231; E-28 to K-231; G-29 to K-231;V-30 to K-231; Q-31 to K-231; I-32 to K-231; Q-33 to K-231; I-34 toK-231; I-35 to K-231; Y-36 to K-231; F-37 to K-231; N-38 to K-231; L-39to K-231; E-40 to K-231; T-41 to K-231; V-42 to K-231; Q-43 to K-231;V-44 to K-231; T-45 to K-231; W-46 to K-231; N-47 to K-231; A-48 toK-231; S-49 to K-231; K-50 to K-231; Y-51 to K-231; S-52 to K-231; R-53to K-231; T-54 to K-231; N-55 to K-231; L-56 to K-231; T-57 to K-231;F-58 to K-231; H-59 to K-231; Y-60 to K-231; R-61 to K-231; F-62 toK-231; N-63 to K-231; G-64 to K-231; D-65 to K-231; E-66 to K-231; A-67to K-231; Y-68 to K-231; D-69 to K-231; Q-70 to K-231; C-71 to K-231;T-72 to K-231; N-73 to K-231; Y-74 to K-231; L-75 to K-231; L-76 toK-231; Q-77 to K-231; E-78 to K-231; G-79 to K-231; H-80 to K-231; T-81to K-231; S-82 to K-231; G-83 to K-231; C-84 to K-231; L-85 to K-231;L-86 to K-231; D-87 to K-231; A-88 to K-231; E-89 to K-231; Q-90 toK-231; R-91 to K-231; D-92 to K-231; D-93 to K-231; I-94 to K-231; L-95to K-231; Y-96 to K-231; F-97 to K-231; S-98 to K-231; I-99 to K-231;R-100 to K-231; N-101 to K-231; G-102 to K-231; T-103 to K-231; H-104 toK-231; P-105 to K-231; V-106 to K-231; F-107 to K-231; T-108 to K-231;A-109 to K-231; S-110 to K-231; R-111 to K-231; W-112 to K-231; M-113 toK-231; V-114 to K-231; Y-115 to K-231; Y-116 to K-231; L-117 to K-231;K-118 to K-231; P-119 to K-231; S-120 to K-231; S-121 to K-231; P-122 toK-231; K-123 to K-231; H-124 to K-231; V-125 to K-231; R-126 to K-231;F-127 to K-231; S-128 to K-231; W-129 to K-231; H-130 to K-231; Q-131 toK-231; D-132 to K-231; A-133 to K-231; V-134 to K-231; T-135 to K-231;V-136 to K-231; T-137 to K-231; C-138 to K-231; S-139 to K-231; D-140 toK-231; L-141 to K-231; S-142 to K-231; Y-143 to K-231; G-144 to K-231;D-145 to K-231; L-146 to K-231; L-147 to K-231; Y-148 to K-231; E-149 toK-231; V-150 to K-231; Q-151 to K-231; Y-152 to K-231; R-153 to K-231;S-154 to K-231; P-155 to K-231; F-156 to K-231; D-157 to K-231; T-158 toK-231; E-159 to K-231; W-160 to K-231; Q-161 to K-231; S-162 to K-231;K-163 to K-231; Q-164 to K-231; E-165 to K-231; N-166 to K-231; T-167 toK-231; C-168 to K-231; N-169 to K-231; V-170 to K-231; T-171 to K-231;I-172 to K-231; E-173 to K-231; G-174 to K-231; L-175 to K-231; D-176 toK-231; A-177 to K-231; E-178 to K-231; K-179 to K-231; C-180 to K-231;Y-181 to K-231; S-182 to K-231; F-183 to K-231; W-184 to K-231; V-185 toK-231; R-186 to K-231; V-187 to K-231; K-188 to K-231; A-189 to K-231;M-190 to K-231; E-191 to K-231; D-192 to K-231; V-193 to K-231; Y-194 toK-231; G-195 to K-231; P-196 to K-231; D-197 to K-231; T-198 to K-231;Y-199 to K-231; P-200 to K-231; S-201 to K-231; D-202 to K-231; W-203 toK-231; S-204 to K-231; E-205 to K-231; V-206 to K-231; T-207 to K-231;C-208 to K-231; W-209 to K-231; Q-210 to K-231; R-211 to K-231; G-212 toK-231; E-213 to K-231; I-214 to K-231; R-215 to K-231; D-216 to K-231;A-217 to K-231; C-218 to K-231; A-219 to K-231; E-220 to K-231; T-221 toK-231; P-222 to K-231; T-223 to K-231; P-224 to K-231; P-225 to K-231;K-226 to K-231; of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Additionally, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of C-terminal deletions of the CRCGCL polypeptideof the invention shown as SEQ ID NO:2 include polypeptides comprisingthe amino acid sequence of residues: Q-23 to K-231; Q-23 to S-230; Q-23to L-229; Q-23 to K-228; Q-23 to P-227; Q-23 to K-226; Q-23 to P-225;Q-23 to P-224; Q-23 to T-223; Q-23 to P-222; Q-23 to T-221; Q-23 toE-220; Q-23 to A-219; Q-23 to C-218; Q-23 to A-217; Q-23 to D-216; Q-23to R-215; Q-23 to I-214; Q-23 to E-213; Q-23 to G-212; Q-23 to R-211;Q-23 to Q-210; Q-23 to W-209; Q-23 to C-208; Q-23 to T-207; Q-23 toV-206; Q-23 to E-205; Q-23 to S-204; Q-23 to W-203; Q-23 to D-202; Q-23to S-201; Q-23 to P-200; Q-23 to Y-199; Q-23 to T-198; Q-23 to D-197;Q-23 to P-196; Q-23 to G-195; Q-23 to Y-194; Q-23 to V-193; Q-23 toD-192; Q-23 to E-191; Q-23 to M-190; Q-23 to A-189; Q-23 to K-188; Q-23to V-187; Q-23 to R-186; Q-23 to V-185; Q-23 to W-184; Q-23 to F-183;Q-23 to S-182; Q-23 to Y-181; Q-23 to C-180; Q-23 to K-179; Q-23 toE-178; Q-23 to A-177; Q-23 to D-176; Q-23 to L-175; Q-23 to G-174; Q-23to E-173; Q-23 to I-172; Q-23 to T-171; Q-23 to V-170; Q-23 to N-169;Q-23 to C-168; Q-23 to T-167; Q-23 to N-166; Q-23 to E-165; Q-23 toQ-164; Q-23 to K-163; Q-23 to S-162; Q-23 to Q-161; Q-23 to W-160; Q-23to E-159; Q-23 to T-158; Q-23 to D-157; Q-23 to F-156; Q-23 to P-155;Q-23 to S-154; Q-23 to R-153; Q-23 to Y-152; Q-23 to Q-151; Q-23 toV-150; Q-23 to E-149; Q-23 to Y-148; Q-23 to L-147; Q-23 to L-146; Q-23to D-145; Q-23 to G-144; Q-23 to Y-143; Q-23 to S-142; Q-23 to L-141;Q-23 to D-140; Q-23 to S-139; Q-23 to C-138; Q-23 to T-137; Q-23 toV-136; Q-23 to T-135; Q-23 to V-134; Q-23 to A-133; Q-23 to D-132; Q-23to Q-131; Q-23 to H-130; Q-23 to W-129; Q-23 to S-128; Q-23 to F-127;Q-23 to R-126; Q-23 to V-125; Q-23 to H-124; Q-23 to K-123; Q-23 toP-122; Q-23 to S-121; Q-23 to S-120; Q-23 to P-119; Q-23 to K-118; Q-23to L-117; Q-23 to Y-116; Q-23 to Y-115; Q-23 to V-114; Q-23 to M-113;Q-23 to W-112; Q-23 to R-111; Q-23 to S-110; Q-23 to A-109; Q-23 toT-108; Q-23 to F-107; Q-23 to V-106; Q-23 to P-105; Q-23 to H-104; Q-23to T-103; Q-23 to G-102; Q-23 to N-101; Q-23 to R-100; Q-23 to I-99;Q-23 to S-98; Q-23 to F-97; Q-23 to Y-96; Q-23 to L-95; Q-23 to I-94;Q-23 to D-93; Q-23 to D-92; Q-23 to R-91; Q-23 to Q-90; Q-23 to E-89;Q-23 to A-88; Q-23 to D-87; Q-23 to L-86; Q-23 to L-85; Q-23 to C-84;Q-23 to G-83; Q-23 to S-82; Q-23 to T-81; Q-23 to H-80; Q-23 to G-79;Q-23 to E-78; Q-23 to Q-77; Q-23 to L-76; Q-23 to L-75; Q-23 to 74; Q-23to N-73; Q-23 to T-72; Q-23 to C-71; Q-23 to Q-70; Q-23 to D-69; Q-23 toY-68; Q-23 to A-67; Q-23 to E-66; Q-23 to D-65; Q-23 to G-64; Q-23 toN-63; Q-23 to F-62; Q-23 to R-61; Q-23 to Y-60; Q-23 to H-59; Q-23 toF-58; Q-23 to T-57; Q-23 to L-56; Q-23 to N-55; Q-23 to T-54; Q-23 toR-53; Q-23 to S-52; Q-23 to Y-51; Q-23 to K-50; Q-23 to S-49; Q-23 toA-48; Q-23 to N-47; Q-23 to W-46; Q-23 to T-45; Q-23 to V-44; Q-23 toQ-43; Q-23 to V-42; Q-23 to T-41; Q-23 to E-40; Q-23 to L-39; Q-23 toN-38; Q-23 to F-37; Q-23 to Y-36; Q-23 to I-35; Q-23 to I-34; Q-23 toQ-33; Q-23 to I-32; Q-23 to Q-31; Q-23 to V-30; Q-23 to G-29; of SEQ IDNO:2. Polynucleotides encoding these polypeptides are also encompassedby the invention.

Moreover, a signal sequence may be added to these C-terminal contructs.For example, amino acids 1-22 of SEQ ID NO:2, amino acids 2-22 of SEQ IDNO:2, amino acids 3-22 of SEQ ID NO:2, amino acids 4-22 of SEQ ID NO:2,amino acids 5-22 of SEQ ID NO:2, amino acids 6-22 of SEQ ID NO:2, aminoacids 7-22 of SEQ ID NO:2, amino acids 8-22 of SEQ ID NO:2, amino acids9-22 of SEQ ID NO:2, amino acids 10-22 of SEQ ID NO:2, amino acids 11-22of SEQ ID NO:2, amino acids 12-22 of SEQ ID NO:2, amino acids 13-22 ofSEQ ID NO:2, amino acids 14-22 of SEQ ID NO:2, amino acids 15-22 of SEQID NO:2, amino acids 16-22 of SEQ ID NO:2, amino acids 17-22 of SEQ IDNO:2, amino acids 18-22 of SEQ ID NO:2, amino acids 19-22 of SEQ IDNO:2, amino acids 20-22 of SEQ ID NO:2, or amino acids 21-22 of SEQ IDNO:2 can be added to the N-terminus of each C-terminal constructs listedabove.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete CRCGCL amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209641 or209691, where this portion excludes any integer of amino acid residuesfrom 1 to about 361 amino acids from the amino terminus of the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 209641 or 209691, or any integer of amino acid residues from 1 toabout 361 amino acids from the carboxy terminus, or any combination ofthe above amino terminal and carboxy terminal deletions, of the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 209641 or 209691. Polynucleotides encoding all of the above deletionmutant polypeptide forms also are provided.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to theCRCGCL polypeptide sequence set forth herein m-n. In preferredembodiments, the application is directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical topolypeptides having the amino acid sequence of the specific CRCGCL N-and C-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of CRCGCL. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) CRCGCL (SEQ IDNO:2). Certain preferred regions are those set out in FIG. 3 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIGS.1A-1B (SEQ ID NO:2), such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of CRCGCL. Preferred embodiments of the inventionin this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of CRCGCL.

The data representing the structural or functional attributes of CRCGCLset forth in FIGS. 1A-1B and/or Table 1, as described above, wasgenerated using the various modules and algorithms of the DNA*STAR seton default parameters. In a preferred embodiment, the data presented incolumns VIII, IX, XIII, and XIV of Table 1 can be used to determineregions of CRCGCL which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table 1, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table 1). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table 1include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.1A-1B. As set out in FIG. 3 and in Table 1, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

TABLE 1 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 . . B . . . . −0.56 0.01 * * . −0.10 0.59 Gly 2 . . B . . . . −0.980.23 * . . −0.10 0.34 Arg 3 . A B . . . . −1.40 0.49 * * . −0.60 0.22Leu 4 . A B . . . . −1.30 0.74 * * . −0.60 0.18 Val 5 . A B . . . .−1.26 1.04 * * . −0.60 0.20 Leu 6 . A B . . . . −1.24 1.04 * * . −0.600.10 Leu 7 A A . . . . . −1.49 1.54 * * . −0.60 0.12 Trp 8 A A . . . . .−2.46 1.36 * * . −0.60 0.17 Gly 9 A A . . . . . −2.34 1.36 . . . −0.600.15 Ala 10 A A . . . . . −2.30 1.46 . . . −0.60 0.16 Ala 11 A A . . . .. −2.30 1.46 . . . −0.60 0.12 Val 12 . A B . . . . −1.83 1.23 . . .−0.60 0.10 Phe 13 . A B . . . . −1.89 1.23 . . . −0.60 0.10 Leu 14 . A B. . . . −1.83 1.16 . . . −0.60 0.10 Leu 15 A . . . . T . −1.84 1.57 . .. −0.20 0.14 Gly 16 . . . . . T C −1.84 1.54 . . . 0.00 0.16 Gly 17 . .. . T T . −1.80 1.26 . . . 0.20 0.19 Trp 18 A . . . . T . −1.44 1.26 . .. −0.20 0.19 Met 19 . . B . . . . −0.63 1.00 * . . −0.40 0.19 Ala 20 . .B . . . . −0.17 0.97 . . . −0.40 0.34 Leu 21 . . B . . . . −0.17 0.97 .. . −0.40 0.32 Gly 22 . . . . . T C −0.41 0.49 . . F 0.32 0.32 Gln 23 .. . . . T C −0.71 0.37 . . F 0.79 0.32 Gly 24 . . . . . T C −0.11 0.37 .. F 0.96 0.39 Gly 25 . . . . . T C 0.13 −0.31 . . F 1.73 0.69 Ala 26 . .. . . . C 0.09 −0.31 . . F 1.70 0.39 Ala 27 A . . . . . . 0.43 −0.07 * .F 1.33 0.29 Glu 28 A . . B . . . −0.46 −0.10 . * . 0.81 0.52 Gly 29 A .. B . . . −0.11 0.16 . * . 0.04 0.36 Val 30 A . . B . . . −0.66 0.06 . *. −0.13 0.61 Gln 31 . . B B . . . −0.96 0.24 . * . −0.30 0.25 Ile 32 . .B B . . . −0.61 0.93 . . . −0.60 0.18 Gln 33 . . B B . . . −1.31 1.26 .. . −0.60 0.37 Ile 34 . . B B . . . −0.97 1.40 . * . −0.60 0.19 Ile 35 .. B B . . . −0.92 1.40 . * . −0.60 0.43 Tyr 36 . . B B . . . −0.92 1.40. * . −0.60 0.20 Phe 37 . . B B . . . −0.34 1.00 * . . −0.60 0.50 Asn 38. . . B . . C −1.20 0.80 * . . −0.25 1.03 Leu 39 . . . B . . C −0.310.76 . . . −0.40 0.49 Glu 40 . . B B . . . −0.28 0.40 . * . −0.60 0.98Thr 41 . . B B . . . −0.34 0.26 . * . −0.30 0.45 Val 42 . . B B . . .0.07 0.34 . * . −0.30 0.79 Gln 43 . . B B . . . 0.07 0.57 . * . −0.600.48 Val 44 . . B B . . . 0.29 0.97 . * . −0.60 0.53 Thr 45 . . B B . .. −0.01 0.99 * * . −0.60 0.73 Trp 46 . . B B . . . 0.34 0.73 * * . −0.600.56 Asn 47 A . . B . . . 0.96 0.33 * * . −0.15 1.51 Ala 48 . . . B T .C 0.66 0.44 * * F 0.44 1.64 Ser 49 . . . . . T C 1.62 0.34 * * F 1.282.10 Lys 50 . . . . T T . 1.62 −0.57 * * F 2.72 2.55 Tyr 51 . . . . T T. 1.91 −0.49 * * F 2.76 3.65 Ser 52 . . . . T T . 1.10 −0.59 * * F 3.404.38 Arg 53 . . . B T . . 1.38 −0.29 * * F 2.36 1.80 Thr 54 . . B B . .. 0.98 0.20 * * F 1.02 1.66 Asn 55 . . B B . . . 0.90 0.23 * * F 0.681.07 Leu 56 . . B B . . . 0.90 0.34 * * . 0.04 0.75 Thr 57 . . B B . . .1.31 1.10 * * . −0.60 0.81 Phe 58 . . B B . . . 0.50 0.61 * * . −0.600.99 His 59 . . B B . . . 0.81 1.00 . * . −0.45 1.04 Tyr 60 . . B B . .. 0.47 0.71 . * . −0.45 1.15 Arg 61 . . B B . . . 1.28 0.66 . * . −0.451.32 Phe 62 . . . B T . . 1.59 −0.13 . * . 1.19 1.62 Asn 63 . . . . T T. 1.70 −0.63 . * F 2.38 1.79 Gly 64 . . . . T T . 1.49 −0.89 * * F 2.570.92 Asp 65 . . . . T T . 1.73 −0.13 . * F 2.76 1.67 Glu 66 . . . . T T. 1.62 −0.91 . * F 3.40 1.73 Ala 67 . . . . T . . 1.66 −0.91 . . F 2.863.04 Tyr 68 . . . . T . . 1.34 −0.77 * . . 2.22 0.97 Asp 69 . . . . T .. 1.69 −0.29 * . F 1.73 0.81 Gln 70 . . . . T . . 1.44 0.11 * . F 0.941.29 Cys 71 . . B . . T . 0.63 0.37 * . F 0.40 1.29 Thr 72 . . B . . T .0.41 0.30 * . F 0.25 0.64 Asn 73 . . B . . T . 0.66 0.99 * . . −0.200.30 Tyr 74 . . B . . T . 0.66 0.99 * . . −0.20 0.98 Leu 75 . . B . . .. 0.31 0.41 * . . −0.25 1.18 Leu 76 . . B . . . . 0.94 0.36 * . . −0.100.73 Gln 77 . . B . . . . 0.94 0.46 * . F 0.00 0.63 Glu 78 . . B . . . .0.64 0.19 * . F 0.70 1.10 Gly 79 . . . . T . . 0.54 −0.11 * . F 1.951.79 His 80 . . . . T . . 0.69 −0.37 . . F 2.20 1.02 Thr 81 . . . . T T. 0.69 −0.20 * . F 2.50 0.32 Ser 82 . . . . T T . −0.12 0.49 . . F 1.350.26 Gly 83 . . . . T T . −0.12 0.74 . . F 1.10 0.16 Cys 84 . . B . . T. −0.37 0.24 . . . 0.60 0.19 Leu 85 . A B . . . . −0.33 0.26 . . . −0.050.14 Leu 86 A A . . . . . −0.02 −0.13 * * . 0.30 0.24 Asp 87 A A . . . .. 0.39 −0.16 * * . 0.30 0.79 Ala 88 A A . . . . . 0.73 −0.73 . * F 0.901.87 Glu 89 A A . . . . . 1.40 −1.41 . * F 0.90 3.79 Gln 90 A . . . . T. 1.32 −2.10 . * F 1.30 3.79 Arg 91 A . . . . T . 1.32 −1.41 . * F 1.302.63 Asp 92 A . . . . T . 1.08 −1.23 . * F 1.30 1.25 Asp 93 A . . . . T. 0.97 −0.47 . * F 1.00 1.13 Ile 94 . . B B . . . 0.67 −0.09 . * . 0.300.50 Leu 95 . . B B . . . −0.22 0.30 . * . −0.30 0.40 Tyr 96 . . B B . .. −0.22 0.99 . * . −0.60 0.17 Phe 97 . . B B . . . −0.22 0.99 * * .−0.60 0.47 Ser 98 . . B B . . . −0.57 0.70 * * . −0.32 0.92 Ile 99 . . B. . T . 0.01 0.44 * * . 0.36 0.58 Arg 100 . . . . T T . 0.79 0.17 * * F1.49 0.97 Asn 101 . . . . T T . 0.82 −0.11 * * F 2.37 0.98 Gly 102 . . .. T T . 0.67 −0.07 * * F 2.80 2.17 Thr 103 . . . B . . C 0.27 −0.11 * *F 1.77 0.82 His 104 . . . B . . C 0.84 0.67 * * F 0.59 0.44 Pro 105 . .B B . . . 0.14 0.76 * . . −0.04 0.65 Val 106 . . B B . . . −0.16 0.83 *. . −0.32 0.45 Phe 107 . . B B . . . 0.30 0.73 * . . −0.60 0.45 Thr 108. . B B . . . 0.32 0.23 * . . −0.30 0.56 Ala 109 . . B B . . . −0.240.71 * . . −0.60 0.80 Ser 110 A . . B . . . −0.89 0.69 * . . −0.60 0.91Arg 111 A . . B . . . −0.28 0.54 * . . −0.60 0.47 Trp 112 . . B B . . .0.18 0.81 * . . −0.60 0.73 Met 113 . . B B . . . −0.32 1.07 * . . −0.600.85 Val 114 . . B B . . . 0.31 1.37 * . . −0.60 0.36 Tyr 115 . . B B .. . 0.40 1.37 * * . −0.60 0.68 Tyr 116 . . B . . . . −0.01 0.89 * * .−0.25 1.07 Leu 117 . . B . . . . −0.02 0.66 . . . 0.05 1.92 Lys 118 . .. . . T C 0.37 0.40 . . F 0.90 1.65 Pro 119 . . . . T T . 1.27 0.07 . .F 1.70 1.62 Ser 120 . . . . . T C 1.48 −0.69 . . F 2.70 3.94 Ser 121 . .. . . T C 0.87 −0.87 * * F 3.00 2.68 Pro 122 . . B . . . . 1.79−0.23 * * F 2.00 1.29 Lys 123 . . B . T . . 1.04 −0.66 * * F 2.40 1.88His 124 . . B . . . . 0.96 −0.26 * * . 1.25 1.21 Val 125 . . B . . . .0.97 −0.26 * * . 0.95 1.05 Arg 126 . . B . . . . 1.23 0.23 * * . −0.100.55 Phe 127 . . B . . . . 1.44 0.73 * * . −0.38 0.55 Ser 128 . . . . T. . 1.40 0.63 * * . 0.19 1.29 Trp 129 . . . . T . . 0.84 −0.01 . * .1.11 1.10 His 130 . . . . . . C 0.84 0.49 . * . 0.03 1.28 Gln 131 . . .B T . . 0.42 0.34 . * . 0.20 0.71 Asp 132 . . . B T . . 0.27 0.44 . . .−0.12 0.98 Ala 133 . . B B . . . 0.26 0.17 . . . −0.24 0.53 Val 134 . .B B . . . −0.12 0.16 . . . −0.26 0.44 Thr 135 . . B B . . . −0.39 0.33 .. . −0.28 0.14 Val 136 . . B B . . . −0.39 0.71 * . . −0.60 0.19 Thr 137. . B B . . . −1.20 0.21 * . . −0.30 0.43 Cys 138 . . B . . T . −0.910.26 . . . 0.10 0.24 Ser 139 . . B . . T . −0.30 0.16 . . F 0.47 0.44Asp 140 . . B . . T . −0.33 0.27 . . F 0.69 0.48 Leu 141 . . B . . T .0.52 0.21 . . . 0.76 0.88 Ser 142 . . . . T T . 0.02 −0.36 . . . 2.131.10 Tyr 143 . . . . T T . −0.12 −0.06 . . . 2.20 0.54 Gly 144 . . . . TT . −0.07 0.63 . . . 1.08 0.54 Asp 145 . . B . . T . −0.07 0.70 . * .0.46 0.63 Leu 146 . A B B . . . −0.11 0.31 * * . 0.14 0.70 Leu 147 . A BB . . . 0.19 0.20 * * . −0.08 0.52 Tyr 148 . A B B . . . 0.19 0.17 * * .−0.30 0.54 Glu 149 . A B B . . . 0.64 0.93 * * . −0.45 1.03 Val 150 . AB B . . . 0.34 0.24 * * . −0.15 2.45 Gln 151 . . B B . . . 0.94 −0.06. * . 0.45 2.10 Tyr 152 . . . . T . . 1.06 −0.39 * * . 1.39 1.87 Arg 153. . B . . . . 1.30 0.40 . * F 0.58 2.19 Ser 154 . . . . . T C 0.99 −0.24. * F 2.22 2.11 Pro 155 . . . . . T C 1.84 −0.16 . * F 2.56 1.94 Phe 156. . . . T T . 1.56 −0.91 * * F 3.40 1.72 Asp 157 . . . . . T C 1.800.00 * * F 1.96 1.35 Thr 158 . . . . . . C 1.39 0.01 . * F 1.76 1.51 Glu159 A . . . . . . 1.73 −0.03 . * F 2.16 2.33 Trp 160 A . . . . T . 1.94−0.81 . * F 2.66 2.80 Gln 161 A . . . . T . 2.64 −0.41 . * F 2.36 3.36Ser 162 . . . . T T . 2.64 −0.90 . * F 3.40 3.36 Lys 163 . . . . T T .2.64 −0.50 . * F 2.76 5.13 Gln 164 . . . . T . . 1.98 −0.93 . * F 2.694.28 Glu 165 . . . . T . . 2.27 −0.76 . * F 2.52 1.71 Asn 166 . . . . TT . 1.41 −0.74 . * F 2.55 1.38 Thr 167 . . . . T T . 1.40 −0.10 . * F1.93 0.59 Cys 168 . . B . . T . 0.47 −0.01 . * F 1.70 0.49 Asn 169 . . B. . T . 0.47 0.67 . * . 0.48 0.21 Val 170 . . B B . . . 0.12 0.27 * * .0.21 0.26 Thr 171 . . B B . . . −0.69 0.21 * * . 0.04 0.47 Ile 172 . A BB . . . −0.38 0.33 * * . −0.13 0.24 Glu 173 A A . B . . . −0.30 −0.07. * . 0.30 0.55 Gly 174 A A . . . . . −0.30 −0.21 . * F 0.45 0.38 Leu175 A A . . . . . 0.60 −0.70 . . . 0.60 0.95 Asp 176 A A . . . . . 0.24−1.39 . . F 0.90 1.09 Ala 177 A A . . . . . 0.89 −0.81 . . F 0.75 0.59Glu 178 A A . . . . . 0.59 −0.49 . . F 0.60 1.13 Lys 179 A A . B . . .0.23 −0.79 . . . 0.60 0.90 Cys 180 A A . B . . . 0.76 0.00 . . . −0.300.77 Tyr 181 A A . B . . . −0.10 0.41 * * . −0.60 0.47 Ser 182 A . . B .. . 0.60 1.06 * * . −0.60 0.17 Phe 183 A . . B . . . −0.26 1.06 * * .−0.60 0.64 Trp 184 A . . B . . . −0.26 1.13 * * . −0.60 0.30 Val 185 A .. B . . . −0.18 0.37 . * . −0.30 0.45 Arg 186 A . . B . . . −0.53 0.49. * . −0.60 0.53 Val 187 A . . B . . . −0.23 0.31 . * . −0.30 0.49 Lys188 A . . B . . . 0.47 −0.60 . * . 0.75 1.15 Ala 189 A A . . . . . −0.10−1.24 . * . 0.60 0.98 Met 190 A A . . . . . 0.51 −0.60 . * . 0.60 0.98Glu 191 . A B . . . . 0.06 −0.49 . * . 0.30 0.77 Asp 192 . A B . . . .0.70 −0.06 * * . 0.30 0.76 Val 193 . A B . . . . 0.66 −0.13 * . . 0.451.18 Tyr 194 . A B . . . . 0.93 −0.74 . . . 0.75 1.14 Gly 195 . . B . .T . 1.29 −0.26 . . F 0.85 0.98 Pro 196 . . . . T T . 1.08 0.50 . . F0.50 2.08 Asp 197 . . . . T T . 0.78 0.29 . . F 0.80 2.05 Thr 198 . . .. . T C 1.63 −0.09 . . F 1.48 2.77 Tyr 199 . . B . . . . 1.59 −0.51 . .F 1.66 3.00 Pro 200 . . . . . T C 1.63 −0.03 * . F 2.04 1.89 Ser 201 . .. . T T . 1.84 0.36 * . F 1.92 1.75 Asp 202 . . . . T T . 0.99 −0.13 * .F 2.80 1.94 Trp 203 . . . . T T . 0.99 −0.24 . . F 2.37 0.93 Ser 204 . .B B . . . 0.57 −0.19 . . F 1.44 1.00 Glu 205 . . B B . . . 0.49 0.00 . .. 0.26 0.32 Val 206 . . B B . . . 0.79 0.91 * . . −0.32 0.32 Thr 207 . .B B . . . 0.90 0.40 * . . −0.60 0.42 Cys 208 . . . B T . . 0.84 0.01 * *. 0.10 0.47 Trp 209 . . . . T T . 1.14 0.44 . * . 0.20 0.63 Gln 210 A .. . . T . 0.26 −0.20 * * . 0.70 0.75 Arg 211 . . . . T T . 1.22 0.00 * *F 0.65 0.98 Gly 212 . . . . T T . 1.53 −0.57 * * F 1.70 1.83 Gln 213 . A. . T . . 1.61 −1.49 * * F 1.30 1.76 Ile 214 . A . . T . . 1.23−1.39 * * F 1.15 0.91 Arg 215 . A . . T . . 0.64 −0.81 . * F 1.15 0.49Asp 216 . A . . T . . 0.53 −0.74 * * F 1.15 0.29 Ala 217 . A . . T . .0.57 −0.74 * * . 1.00 0.71 Cys 218 . A B . . . . 0.36 −0.94 * * . 0.600.52 Ala 219 . A . . T . . 0.93 −0.51 * . . 1.00 0.48 Glu 220 . A . . T. . 0.61 −0.03 . . F 0.85 0.69 Thr 221 . A . . . . C 0.40 −0.10 . * F1.14 2.00 Pro 222 . A . . T . . 1.03 −0.24 . . F 1.68 3.06 Thr 223 . . .. . . C 1.49 −0.74 . * F 2.32 3.53 Pro 224 . . . . . T C 2.12 −0.31 . *F 2.56 3.78 Pro 225 . . . . T T . 1.31 −0.80 . * F 3.40 4.89 Lys 226 . .. . . T C 1.32 −0.54 . * F 2.86 2.80 Pro 227 A . . . . T . 1.58 −0.64 *. F 2.32 2.42 Lys 228 A . . . . . . 1.19 −1.07 * * F 1.78 3.13 Leu 229 A. . B . . . 0.51 −0.71 * * F 1.24 1.36 Ser 230 . . B B . . . −0.09 −0.03. * F 0.45 0.62 Lys 231 . . B B . . . −1.02 0.23 . * . −0.30 0.25 Phe232 . . B B . . . −1.11 0.91 * . . −0.60 0.22 Ile 233 . . B B . . .−1.46 0.61 . . . −0.60 0.22 Leu 234 . . B B . . . −1.46 0.61 . . . −0.600.14 Ile 235 . . B B . . . −1.74 1.30 . . . −0.60 0.14 Ser 236 A . . B .. . −2.68 1.01 . . . −0.60 0.20 Ser 237 A . . B . . . −2.79 1.01 . . .−0.60 0.17 Leu 238 A . . B . . . −2.71 1.01 . . . −0.60 0.20 Ala 239 A .. B . . . −2.50 1.01 . . . −0.60 0.12 Ile 240 A . . B . . . −2.47 1.24 .. . −0.60 0.09 Leu 241 A . . B . . . −2.47 1.50 . * . −0.60 0.08 Leu 242A . . B . . . −2.98 1.20 . . . −0.60 0.11 Met 243 . . B B . . . −2.981.39 . * . −0.60 0.13 Val 244 A . . B . . . −3.20 1.39 . . . −0.60 0.13Ser 245 A . . B . . . −3.12 1.39 . . . −0.60 0.13 Leu 246 A . . B . . .−2.61 1.39 . . . −0.60 0.11 Leu 247 A . . B . . . −2.61 1.16 . . . −0.600.19 Leu 248 A . . B . . . −2.30 1.20 * . . −0.60 0.12 Leu 249 A . . B .. . −1.40 1.73 * . . −0.60 0.15 Ser 250 A . . B . . . −1.91 1.04 * . .−0.60 0.36 Leu 251 A . . B . . . −1.39 1.04 * . . −0.60 0.36 Trp 252 A .. B . . . −0.47 1.27 * . . −0.60 0.46 Lys 253 A . . B . . . −0.510.59 * * . −0.60 0.68 Leu 254 A . . B . . . 0.34 0.84 * * . −0.60 0.61Trp 255 A . . B . . . 0.69 0.16 * . . −0.15 1.16 Arg 256 A . . B . . .0.80 −0.76 * . . 0.75 1.16 Val 257 A . . B . . . 0.28 0.03 * . . −0.151.21 Lys 258 . . B B . . . −0.66 0.03 * . . −0.30 0.95 Lys 259 . . B B .. . −0.06 −0.20 * . . 0.30 0.34 Phe 260 . . B B . . . −0.07 0.23 * * .−0.30 0.71 Leu 261 . . B B . . . −1.03 −0.03 * * . 0.30 0.48 Ile 262 . .B B . . . −0.39 0.61 * . . −0.60 0.18 Pro 263 . . B . . . . −0.43 1.04 .. . −0.10 0.32 Ser 264 . . B . . . . −0.69 0.26 * * F 0.65 0.64 Val 265. . . . . . C 0.06 0.00 . . F 1.30 1.41 Pro 266 . . . . . . C 0.57 −0.69. . F 2.50 1.82 Asp 267 . . . . . T C 0.57 −0.73 . . F 3.00 1.82 Pro 268. . . . T T . 0.08 −0.43 . . F 2.60 1.72 Lys 269 . . . . T T . 0.17−0.29 . . F 2.15 0.96 Ser 270 . . B . . T . 0.68 −0.29 * . F 1.45 0.89Ile 271 . . B . . . . 0.08 0.14 * . . 0.20 0.57 Phe 272 . . B . . T .−0.62 0.40 * . . −0.20 0.24 Pro 273 . . B . . T . −0.41 1.19 * * . −0.200.15 Gly 274 . . . . . T C −1.34 0.80 * * . 0.00 0.38 Len 275 . . B . .T . −1.08 0.80 * . . −0.20 0.30 Phe 276 . . B . . . . −0.19 0.51 * . .−0.40 0.27 Gln 277 A . . . . . . 0.17 0.49 . * .−0.40 0.47 Ile 278 A . .. . . . 0.38 0.49 . . . −0.40 0.56 His 279 A . . . . T . 0.02 0.20 * * .0.25 1.04 Gln 280 . . . . . T C 0.83 0.20 * . F 0.45 0.52 Gly 281 . . .. . T C 1.53 0.60 * . F 0.30 1.29 Asn 282 . . . . . T C 1.24 −0.09 . . F1.20 1.64 Phe 283 . A . . . . C 1.24 0.33 . . F 0.05 1.00 Gln 284 . A .. . . C 0.97 0.61 * * . −0.16 0.71 Gln 285 . A B . . . . 0.97 0.67 * * .−0.12 0.63 Trp 286 . A B . . . . 1.00 0.27 * * . 0.57 1.22 Ile 287 . A B. . . . 1.00 −0.03 * . . 1.41 1.02 Thr 288 . . . . T . . 1.70 −0.03 * .F 2.40 1.02 Asp 289 . . . . T . . 0.84 0.37 * . F 1.56 1.56 Thr 290 A .. . . . . 0.26 0.10 * . F 0.92 1.65 Gln 291 A A . . . . . 0.51 −0.09 * .F 1.08 1.15 Asn 292 A A . . . . . 0.59 −0.07 . . F 0.69 0.94 Val 293 A A. . . . . 0.87 0.61 * . . −0.60 0.54 Ala 294 A A . . . . . 0.91 0.63 * .. −0.60 0.42 His 295 A A . . . . . 0.62 0.23 * . . −0.30 0.52 Leu 296 AA . . . . . 0.03 0.44 * . . −0.60 0.70 His 297 A A . . . . . −0.310.30 * . . −0.30 0.70 Lys 298 A A . . . . . −0.04 0.23 * . . −0.30 0.51Met 299 A A . . . . . 0.54 0.23 . . . −0.30 0.62 Ala 300 A A . . . . .0.58 −0.46 * . . 0.30 0.79 Gly 301 A A . . . . . 1.39 −0.56 * . . 0.600.69 Ala 302 A A . . . . . 1.12 −0.56 . . F 0.90 1.20 Glu 303 A A . . .. . 0.73 −0.79 . . F 1.20 1.60 Gln 304 A A . . . . . 1.12 −0.86 . . F1.50 1.60 Glu 305 . A . . . . C 1.71 −0.86 . . F 2.00 2.44 Ser 306 . A .. . . C 2.06 −1.36 . . F 2.30 2.44 Gly 307 . . . . . T C 2.43 −1.36 . .F 3.00 2.44 Pro 308 . . . . . T C 1.62 −1.33 . . F 2.70 2.18 Glu 309 . .. . . T C 0.77 −0.64 . . F 2.40 1.34 Glu 310 A . . . . T . −0.09 −0.39 .. F 1.60 1.01 Pro 311 A . . . . . . 0.21 −0.17 . . F 0.95 0.48 Leu 312 AA . . . . . −0.26 −0.20 . . . 0.30 0.48 Val 313 A A . . . . . −0.630.49 * . . −0.60 0.23 Val 314 A A . . . . . −0.59 0.99 * . . −0.60 0.15Gln 315 A A . . . . . −0.90 0.56 * . . −0.60 0.36 Leu 316 A A . . . . .−0.69 0.36 * . . −0.30 0.71 Ala 317 A A . . . . . −0.47 −0.29 * * . 0.451.65 Lys 318 A A . . . . . 0.39 −0.43 * . F 0.45 0.96 Thr 319 A A . . .. . 0.94 −0.83 * . F 0.90 2.03 Glu 320 A A . . . . . 0.73 −1.13 * . F0.90 2.69 Ala 321 A A . . . . . 1.66 −1.20 * . F 1.24 2.08 Glu 322 A A .. . . . 1.64 −1.20 * * F 1.58 2.82 Ser 323 A . . . . T . 0.79 −1.07 * .F 2.32 1.61 Pro 324 A . . . . T . 1.10 −0.39 * . F 2.36 1.32 Arg 325 . .. . T T . 0.89 −0.89 * . F 3.40 1.27 Met 326 A . . . . T . 1.48 −0.46 *. . 2.21 1.46 Leu 327 A . . . . . . 1.17 −0.44 * . F 1.82 1.64 Asp 328 A. . . . T . 1.47 −0.39 * . F 1.68 1.21 Pro 329 A . . . . T . 1.68−0.39 * * F 1.34 2.11 Gln 330 A . . . . T . 1.61 −1.00 * . F 1.30 4.44Thr 331 A . . . . T . 2.21 −1.69 . * F 1.30 5.31 Glu 332 A A . . . . .2.43 −1.69 . * F 0.90 5.95 Glu 333 A A . . . . . 2.13 −1.61 . . F 0.903.47 Lys 334 A A . . . . . 2.00 −1.63 . . F 1.15 3.22 Glu 335 A A . . .. . 1.66 −1.69 . . F 1.40 1.84 Ala 336 A . . . . T . 1.67 −1.26 . . F2.05 1.05 Ser 337 A . . . . T . 0.86 −0.87 . . F 2.15 0.71 Gly 338 . . .. T T . 0.86 −0.19 . * F 2.50 0.34 Gly 339 . . . . T T . −0.00 0.21 * .F 1.65 0.58 Ser 340 . . . . . . C −0.21 0.40 . . F 0.70 0.35 Leu 341 . .. . . . C. 0.34 0.44 . * . 0.30 0.55 Gln 342 . . B . . . . 0.64 0.51 . *. −0.15 0.76 Leu 343 . . B . . . . 0.78 0.49 . * . −0.40 0.98 Pro 344 .. B . . . . 0.31 0.53 * * . −0.25 1.84 His 345 . . B . . . . 0.610.53 * * . −0.40 0.88 Gln 346 . . B . . . . 1.08 0.53 * . F 0.03 1.84Pro 347 . . B . . . . 0.73 0.27 * . F 0.46 1.18 Leu 348 . . . . T T .1.54 0.27 * . F 1.04 0.86 Gln 349 . . . . T T . 0.90 −0.23 * . F 1.770.83 Gly 350 . . . . T T . 0.08 0.01 * . F 1.30 0.40 Gly 351 . . B . . T. −0.23 0.23 . . F 0.77 0.36 Asp 352 . . B B . . . −0.91 0.03 . . F 0.240.30 Val 353 . . B B . . . −0.44 0.31 . . F 0.11 0.21 Val 354 . . B B .. . −0.79 0.31 . . . −0.17 0.21 Thr 355 . . B B . . . −1.14 0.31 . . .−0.30 0.13 Ile 356 . . B B . . . −1.11 1.10 . . . −0.60 0.15 Gly 357 . .B B . . . −1.81 0.94 . . . −0.60 0.28 Gly 358 . . B B . . . −1.81 1.09 .. . −0.60 0.17 Phe 359 . . B B . . . −1.56 1.24 . . . −0.60 0.18 Thr 360. . B B . . . −1.24 1.17 . . . −0.60 0.18 Phe 361 . . B B . . . −0.361.14 . * . −0.60 0.29 Val 362 . . B B . . . 0.10 0.71 . * . −0.32 0.57Met 363 . . B B . . . 0.14 −0.07 . * . 0.86 0.77 Asn 364 . . . B T . .0.60 −0.17 . * . 1.69 1.19 Asp 365 . . . . T T . 0.06 −0.20 . * . 2.372.51 Arg 366 . . . . T T . 0.17 −0.20 . * F 2.80 1.88 Ser 367 A . . . .T . 0.21 −0.31 . * . 1.97 1.18 Tyr 368 A . . . . T . 0.42 −0.03 . * .1.54 0.58 Val 369 . A B . . . . 0.03 0.40 . * . −0.04 0.38 Ala 370 . A B. . . . −0.36 0.83 * . . −0.32 0.36 Leu 371 . A B . . . . −0.86 0.87 * *. −0.60 0.30 Ter 372 . A B . . . . −0.94 0.54 . . . −0.60 0.51

Among highly preferred fragments in this regard are those that compriseregions of CRCGCL that combine several structural features, such asseveral of the features set out above.

Other preferred fragments are biologically active CRCGCL fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the CRCGCL polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

However, many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.One embodiment of the present invention excludes Genbank Accession No.X91553 (herein incorporated by reference in its entirety.) Moreover,preferably excluded from the present invention are one or morepolynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1559 of SEQID NO:1, b is an integer of 15 to 1573, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:1, and where theb is greater than or equal to a+14.

Epitope-bearing Portions

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the polypeptides of the presentinvention. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 81:3998-4002.However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al. (1995) Mol. Gen. Genet.249:425-431. Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

A list of exemplified amino acid sequences comprising immunogenicepitopes are shown in Table 1 below. It is pointed out that Table 1 onlylists amino acid residues comprising epitopes predicted to have thehighest degree of antigenicity using the algorithm of Jameson and Wolf,(1988) Comp. Appl. Biosci. 4:181-186 (said references incorporated byreference in their entireties). The Jameson-Wolf antigenic analysis wasperformed using the computer program PROTEAN, using default parameters(Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South ParkStreet Madison, Wis.). Table 1 and portions of polypeptides not listedin Table 1 are not considered non-immunogenic. The immunogenic epitopesof Table 1 is an exemplified list, not an exhaustive list, because otherimmunogenic epitopes are merely not recognized as such by the particularalgorithm used. Amino acid residues comprising other immunogenicepitopes may be routinely determined using algorithms similar to theJameson-Wolf analysis or by in vivo testing for an antigenic responseusing methods known in the art. See, e.g., Geysen et al., supra; U.S.Pat. Nos. 4,708,781; 5,194,392; 4,433,092; and 5,480,971 (saidreferences incorporated by reference in their entireties).

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples antigenic polypeptides or peptides thatcan be used to CRCGCL -specific antibodies include: 22-29; 48-56; 62-73;78-85; 88-95; 99-105; 118-126; 139-146; 151-169; 188-206; 208-231;264-271; 286-293; 300-313; 317-342; 347-353; 363-369 amino acid residuesof SEQ ID NO:2 These polypeptide fragments have been determined to bearantigenic epitopes of the CRCGCL protein by the analysis of theJameson-Wolf antigenic index, as shown in FIG. 3, above.

It is particularly pointed out that the amino acid sequences of Table 1comprise immunogenic epitopes. Table 1 lists only the critical residuesof immunogenic epitopes determined by the Jameson-Wolf analysis. Thus,additional flanking residues on either the N-terminal, C-terminal, orboth N- and C-terminal ends may be added to the sequences of Table 1 togenerate an epitope-bearing polypeptide of the present invention.Therefore, the immunogenic epitopes of Table 1 may include additionalN-terminal or C-terminal amino acid residues. The additional flankingamino acid residues may be contiguous flanking N-terminal and/orC-terminal sequences from the polypeptides of the present invention,heterologous polypeptide sequences, or may include both contiguousflanking sequences from the polypeptides of the present invention andheterologous polypeptide sequences.

Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

The immunogenic and antigenic epitope-bearing fragments may be specifiedby either the number of contiguous amino acid residues, as describedabove, or further specified by N-terminal and C-terminal positions ofthese fragments on the amino acid sequence of SEQ ID NO:2. Everycombination of a N-terminal and C-terminal position that a fragment of,for example, at least 7 or at least 15 contiguous amino acid residues inlength could occupy on the amino acid sequence of SEQ ID NO:2 isincluded in the invention. Again, “at least 7 contiguous amino acidresidues in length” means 7 amino acid residues in length or any integerbetween 7 amino acids and the number of amino acid residues of the fulllength polypeptide of the present invention. Specifically, each andevery integer between 7 and the number of amino acid residues of thefull length polypeptide are included in the present invention.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If in vivoimmunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)Nature 331:84-86. Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al. (1995) J.Biochem. 270:3958-3964. Nucleic acids encoding the above epitopes canalso be recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes monoclonal, polyclonal, chimeric, humanized,and human monoclonal and polyclonal antibodies which specifically bindthe polypeptides of the present invention. The present invention furtherincludes antibodies which are anti-idiotypic to the antibodies of thepresent invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and FiguresAntibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Monoclonal antibodies can be prepared using a wide varietyof techniques known in the art including the use of hybridoma,recombinant, and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlowet al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). Fab and F(ab′)2fragments may be produced by proteolytic cleavage, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA and phage display technologyor through synthetic chemistry using methods known in the art. Forexample, the antibodies of the present invention can be prepared usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface of aphage particle which carries polynucleotide sequences encoding them.Phage with a desired binding property are selected from a repertoire orcombinatorial antibody library (e.g. human or murine) by selectingdirectly with antigen, typically antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 with Fab, Fv or disulfide stabilized Fvantibody domains recombinantly fused to either the phage gene III orgene VIII protein. Examples of phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman U. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. etal. (1995) J. Immunol. Methods 184:177-186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene 1879-18; Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864-869; and Sawai, H. et al. (1995) AJRI 34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988) Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805-814; Roguska M. A. et al. (1994)PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741 (said references incorporated by reference in theirentireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994)Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies, S. O. et al.(1992) PNAS 89:1428-1432; Fell, H. P. et al. (1991) J. Immunol.146:2446-2452 (said references incorporated by reference in theirentireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. Antibodieswhich act as agonists or antagonists of the polypeptides of the presentinvention include, for example, antibodies which disrupt receptor/ligandinteractions with the polypeptides of the invention either partially orfully. For example, the present invention includes antibodies thatdisrupt the ability of the proteins of the invention to multimerize. Inanother example, the present invention includes antibodies which allowthe proteins of the invention to multimerize, but disrupts the abilityof the proteins of the invention to bind one or more CRCGCLreceptor(s)/ligand(s). In yet another example, the present inventionincludes antibodies which allow the proteins of the invention tomultimerize, and bind CRCGCL receptor(s)/ligand(s), but blocksbiological activity associated with the CRCGCL/receptor/ligand complex.

Antibodies which act as agonists or antagonists of the polypeptides ofthe present invention also include, both receptor-specific antibodiesand ligand-specific antibodies. Included are receptor-specificantibodies that do not prevent ligand binding but prevent receptoractivation. Receptor activation (i.e., signaling) may be determined bytechniques described herein or otherwise known in the art. Also includedare receptor-specific antibodies which both prevent ligand binding andreceptor activation. Likewise, included are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included are antibodies that activate the receptor. Theseantibodies may act as agonists for either all or less than all of thebiological activities affected by ligand-mediated receptor activation.The antibodies may be specified as agonists or antagonists forbiological activities comprising specific activities disclosed herein.The above antibody agonists can be made using methods known in the art.See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res. 58(16):3668-3678(1998); Harrop, J. A. et al., J. Immunol. 161(4):1786-1794 (1998); Zhu,Z. et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, D. Y. et al., J.Immunol. 160(7): 3170-3179 (1998); Prat, M. et al., J. Cell. Sci.111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods205(2):177-190 (1997); Liautard, J. et al., Cytokinde 9(4):233-241(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301(1997); Taryman, R. E. et al., Neuron 14(4):755-762 (1995); Muller, Y.A. et al., Structure 6(9): 1153-1167 (1998); Bartunek, P. et al.,Cytokine 8(1):14-20 (1996)(said references incorporated by reference intheir entireties).

As discussed above, antibodies to the CRCGCL proteins of the inventioncan, in turn, be utilized to generate anti-idiotype antibodies that“mimic” CRCGCL using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) andNissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodieswhich bind to CRCGCL and competitively inhibit CRCGCL multimerizationand/or binding to ligand can be used to generate anti-idiotypes that“mimic” the CRCGCL mutimerization and/or binding domain and, as aconsequence, bind to and neutralize CRCGCL and/or its ligand. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in therapeutic regimens to neutralize CRCGCL ligand. Forexample, such anti-idiotypic antibodies can be used to bind CRCGCL, orto bind CRCGCL ligands/receptors, and thereby block CRCGCL biologicalactivity.

Fusion Proteins

Any CRCGCL polypeptide can be used to generate fusion proteins. Forexample, the CRCGCL polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the CRCGCLpolypeptide can be used to indirectly detect the second protein bybinding to the CRCGCL. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the CRCGCL polypeptidescan be used as a targeting molecule once fused to other proteins.

Examples of domains that can be fused to CRCGCL polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

In certain preferred embodiments, CRCGCL proteins of the inventioncomprise fusion proteins wherein the CRCGCL polypeptides are thosedescribed above as m-n. In preferred embodiments, the application isdirected to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or99% identical to the nucleic acid sequences encoding polypeptides havingthe amino acid sequence of the specific N- and C-terminal deletionsrecited herein. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the CRCGCL polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the CRCGCL polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the CRCGCLpolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the CRCGCL polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

Moreover, CRCGCL polypeptides, including fragments, and specificallyepitopes, can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the CRCGCL polypeptides can be fused to marker sequences, suchas a peptide which facilitates purification of CRCGCL. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the CRCGCLpolynucleotides or the polypeptides.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the CRCGCLpolynucleotide, host cells, and the production of polypeptides byrecombinant techniques. The vector may be, for example, a phage,plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

CRCGCL polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The CRCGCL polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that CRCGCL polypeptidesmay in fact be expressed by a host cell lacking a recombinant vector.

CRCGCL polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

CRCGCL polypeptides, and preferably the secreted form, can also berecovered from: products purified from natural sources, including bodilyfluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells. Depending upon the host employed in a recombinant productionprocedure, the CRCGCL polypeptides may be glycosylated or may benon-glycosylated. In addition, CRCGCL polypeptides may also include aninitial modified methionine residue, in some cases as a result ofhost-mediated processes. Thus, it is well known in the art that theN-terminal methionine encoded by the translation initiation codongenerally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., CRCGCL coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with CRCGCL polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous CRCGCLpolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous CRCGCL polynucleotide sequences via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). For example, apeptide corresponding to a fragment of the CRCGCL polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the CRCGCLpolynucleotide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses CRCGCL polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄,; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofCRCGCL which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The CRCGCL polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of the CRCGCLpolypeptides of the invention, their preparation, and compositions(preferably, pharmaceutical compositions) containing them. In specificembodiments, the polypeptides of the invention are monomers, dimers,trimers or tetramers. In additional embodiments, the multimers of theinvention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyCRCGCL polypeptides of the invention (including CRCGCL fragments,variants, splice variants, and fusion proteins, as described herein).These homomers may contain CRCGCL polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only CRCGCL polypeptides havingan identical amino acid sequence. In another specific embodiment, ahomomer of the invention is a multimer containing CRCGCL polypeptideshaving different amino acid sequences. In specific embodiments, themultimer of the invention is a homodimer (e.g., containing CRCGCLpolypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing CRCGCL polypeptides having identical and/ordifferent amino acid sequences). In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the CRCGCL polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the CRCGCL polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptide encoded by the cloneHTAEK53). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a CRCGCL fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a CRCGCL-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another Cytokine Receptor familymember that is capable of forming covalently associated multimers, suchas for example, oseteoprotegerin (see, e.g., International PublicationNo. WO 98/49305, the contents of which are herein incorporated byreference in its entirety).

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Uses of the CRCGCL Polynucleotides

The CRCGCL polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

There exists an ongoing need to identify new chromosome markers, sincefew chromosome marking reagents, based on actual sequence data (repeatpolymorphisms), are presently available. Using a panel of radiationhybrids, CRCGCL maps to the pseudoautosomal region (PAR) of the sexchromosomes, which is located on both X (Xp22.3) and Y (Yp13.3).Interestingly, two other cytokine receptors map to this region (IL3Ra,and GMCSFRa). See, Kremer et al. “A Cytokine Receptor Gene Cluster inthe X-Y pseudoautosomal region?” Blood 82(1) 22-28 (1993). Thus, CRCGCLpolynucleotides can be used in linkage analysis as a marker for thepseudoautosomal region on the X and Y chromosomes.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:1. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human CRCGCL genecorresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the CRCGCL polynucleotides can be achieved withpanels of specific chromosome fragments. Other gene mapping strategiesthat can be used include in situ hybridization, prescreening withlabeled flow-sorted chromosomes, and preselection by hybridization toconstruct chromosome specific-cDNA libraries.

Precise chromosomal location of the CRCGCL polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the CRCGCL polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in the CRCGCLpolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the CRCGCL polypeptide and the corresponding genefrom several normal individuals is required to distinguish the mutationfrom a polymorphism. If a new polymorphism is identified, thispolymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingCRCGCL polynucleotides. Any of these alterations (altered expression,chromosomal rearrangement, or mutation) can be used as a diagnostic orprognostic marker.

In addition to the foregoing, a CRCGCL polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Both methods rely on binding of the polynucleotide to DNA orRNA. For these techniques, preferred polynucleotides are usually 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

CRCGCL polynucleotides are also useful in gene therapy. One goal of genetherapy is to insert a normal gene into an organism having a defectivegene, in an effort to correct the genetic defect. CRCGCL offers a meansof targeting such genetic defects in a highly accurate manner. Anothergoal is to insert a new gene that was not present in the host genome,thereby producing a new trait in the host cell.

The CRCGCL polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The CRCGCL polynucleotides can beused as additional DNA markers for RFLP.

The CRCGCL polynucleotides can also be used as an alternative to RFLP,by determining the actual base-by-base DNA sequence of selected portionsof an individual's genome. These sequences can be used to prepare PCRprimers for amplifying and isolating such selected DNA, which can thenbe sequenced. Using this technique, individuals can be identifiedbecause each individual will have a unique set of DNA sequences. Once anunique ID database is established for an individual, positiveidentification of that individual, living or dead, can be made fromextremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, etc., can be amplified using PCR. In oneprior art technique, gene sequences amplified from polymorphic loci,such as DQa class II HLA gene, are used in forensic biology to identifyindividuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Oncethese specific polymorphic loci are amplified, they are digested withone or more restriction enzymes, yielding an identifying set of bands ona Southern blot probed with DNA corresponding to the DQa class II HLAgene. Similarly, CRCGCL polynucleotides can be used as polymorphicmarkers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from CRCGCL sequences. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.

Because CRCGCL is found expressed in a cervical cancer cell line (HeLa),activated T cells, and a lung carcinoma cell line (A549), while ashorter variant is also expressed in the lymph node and to a lesserextent in the spleen, CRCGCL polynucleotides are useful as hybridizationprobes for differential identification of the tissue(s) or cell type(s)present in a biological sample. Similarly, polypeptides and antibodiesdirected to CRCGCL polypeptides are useful to provide immunologicalprobes for differential identification of the tissue(s) or cell type(s).In addition, for a number of disorders of the above tissues or cells,particularly of the immune system, significantly higher or lower levelsof CRCGCL gene expression may be detected in certain tissues (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” CRCGCL gene expression level,i.e., the CRCGCL expression level in healthy tissue from an individualnot having the immune system disorder.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves: (a) assaying CRCGCL gene expression level in cells or bodyfluid of an individual; (b) comparing the CRCGCL gene expression levelwith a standard CRCGCL gene expression level, whereby an increase ordecrease in the assayed CRCGCL gene expression level compared to thestandard expression level is indicative of disorder in the immunesystem.

In the very least, the CRCGCL polynucleotides can be used as molecularweight markers on Southern gels, as diagnostic probes for the presenceof a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other support, to raise anti-DNA antibodies using DNAimmunization techniques, and as an antigen to elicit an immune response.

Uses of CRCGCL Polypeptides

CRCGCL polypeptides can be used in numerous ways. The followingdescription should be considered exemplary and utilizes knowntechniques.

CRCGCL polypeptides can be used to assay protein levels in a biologicalsample using antibody-based techniques. For example, protein expressionin tissues can be studied with classical immunohistological methods.(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell . Biol. 105:3087-3096 (1987).) Other antibody-basedmethods useful for detecting protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S),tritium (3H), indium (112In), and technetium (99mTc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of CRCGCL polypeptide in cells orbody fluid of an individual; (b) comparing the level of gene expressionwith a standard gene expression level, whereby an increase or decreasein the assayed CRCGCL polypeptide gene expression level compared to thestandard expression level is indicative of a disorder.

Moreover, CRCGCL polypeptides can be used to treat disease. For example,patients can be administered CRCGCL polypeptides in an effort to replaceabsent or decreased levels of the CRCGCL polypeptide (e.g., insulin), tosupplement absent or decreased levels of a different polypeptide (e.g.,hemoglobin S for hemoglobin B), to inhibit the activity of a polypeptide(e.g., an oncogene), to activate the activity of a polypeptide (e.g., bybinding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth).

Similarly, antibodies directed to CRCGCL polypeptides can also be usedto treat disease. For example, administration of an antibody directed toa CRCGCL polypeptide can bind and reduce overproduction of thepolypeptide. Similarly, administration of an antibody can activate thepolypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

At the very least, the CRCGCL polypeptides can be used as molecularweight markers on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art. CRCGCLpolypeptides can also be used to raise antibodies, which in turn areused to measure protein expression from a recombinant cell, as a way ofassessing transformation of the host cell. Moreover, CRCGCL polypeptidescan be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of the CRCGCLpolypeptide of the present invention. This method requires apolynucleotide that codes for a CRCGCL polypeptide operatively linked toa promoter and any other genetic elements necessary for the expressionof the polypeptide by the target tissue. Such gene therapy and deliverytechniques are known in the art, see, for example, WO90/11092, which isherein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aCRCGCL polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells that are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the CRCGCL polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The CRCGCLpolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the CRCGCL polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the CRCGCL polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The CRCGCL polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of CRCGCL DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forCRCGCL.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The CRCGCL polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked acid sequence injection, an effective dosage amount of DNAor RNA will be in the range of from about 0.05 mg/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked CRCGCL DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

As is evidenced in the Examples, naked CRCGCL nucleic acid sequences canbe administered in vivo results in the successful expression of CRCGCLpolypeptide in the femoral arteries of rabbits.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the CRCGCL polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are be engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding CRCGCL. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding CRCGCL. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express CRCGCL.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with CRCGCL polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses CRCGCL, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The CRCGCL polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe CRCGCL polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the CRCGCL polynucleotide construct integrated intoits genome, and will express CRCGCL.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding CRCGCL) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the CRCGCL desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous CRCGCL sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous CRCGCL sequence.

The polynucleotides encoding CRCGCL may be administered along with otherpolynucleotides encoding other angiongenic proteins. Angiogenic proteinsinclude, but are not limited to, acidic and basic fibroblast growthfactors, VEGF-1, epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding CRCGCL contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities of CRCGCL

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, can be used in assays to test for one or more biologicalactivities. If CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, do exhibit activity in a particular assay, it islikely that CRCGCL may be involved in the diseases associated with thebiological activity. Therefore, CRCGCL could be used to treat theassociated disease.

Immune Activity

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may be useful in treating deficiencies or disorders of theimmune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through a process called hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune deficiencies or disorders may be genetic, somatic, such ascancer or some autoimmune disorders, acquired (e.g., by chemotherapy ortoxins), or infectious. Moreover, CRCGCL polynucleotides orpolypeptides, or agonists or antagonists of CRCGCL, can be used as amarker or detector of a particular immune system disease or disorder.

Interestingly, CRCGCL maps to the pseudoautosomal regions on the X and Ychromosomes. It is likely that mutations in CRCGCL may also lead toimmune disorders, especially those involving activated T cells.Moreover, mutations in CRCGCL may be involved in autoimmune diseases,especially X-linked autoimmune diseases.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may be useful in treating or detecting deficiencies or disordersof hematopoietic cells. CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, could be used to increasedifferentiation and proliferation of hematopoietic cells, including thepluripotent stem cells, in an effort to treat those disorders associatedwith a decrease in certain (or many) types hematopoietic cells. Examplesof immunologic deficiency syndromes include, but are not limited to:blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, CRCGCLpolynucleotides or polypeptides, or agonists or antagonists of CRCGCL,could be used to treat blood coagulation disorders (e.g.,afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.thrombocytopenia), or wounds resulting from trauma, surgery, or othercauses. Alternatively, CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting,important in the treatment of heart attacks (infarction), strokes, orscarring.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may also be useful in treating or detecting autoimmunedisorders. Many autoimmune disorders result from inappropriaterecognition of self as foreign material by immune cells. Thisinappropriate recognition results in an immune response leading to thedestruction of the host tissue. Therefore, the administration of CRCGCLpolynucleotides or polypeptides, or agonists or antagonists of CRCGCL,that can inhibit an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detectedinclude, but are not limited to: Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitis, and autoimmuneinflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL. Moreover, these molecules can be used to treatanaphylaxis, hypersensitivity to an antigenic molecule, or blood groupincompatibility.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may also be used to treat and/or prevent organ rejection orgraft-versus-host disease (GVHD). Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, that inhibits an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in preventing organ rejection orGVHD.

Similarly, CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, may also be used to modulate inflammation. Forexample, CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, may inhibit the proliferation and differentiationof cells involved in an inflammatory response. These molecules can beused to treat inflammatory conditions, both chronic and acuteconditions, including inflammation associated with infection (e.g.,septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, can be used to treat or detect hyperproliferative disorders,including neoplasms. CRCGCL polynucleotides or polypeptides, or agonistsor antagonists of CRCGCL, may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, CRCGCLpolynucleotides or polypeptides, or agonists or antagonists of CRCGCL,may proliferate other cells which can inhibit the hyperproliferativedisorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, include, but are not limited to neoplasms located in the:abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL. Examples of such hyperproliferative disordersinclude, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other hyperproliferative disease, besidesneoplasia, located in an organ system listed above.

Cardiovascular Disorders

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, encoding CRCGCL may be used to treat cardiovascular disorders,including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, are especially effective for the treatment of critical limbischemia and coronary disease. As shown in the Examples, administrationof CRCGCL polynucleotides and polypeptides to an experimentally inducedischemia rabbit hindlimb may restore blood pressure ratio, blood flow,angiographic score, and capillary density.

CRCGCL polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. CRCGCL polypeptides may be administered as part of apharmaceutical composition, described in more detail below. Methods ofdelivering CRCGCL polynucleotides are described in more detail herein.

Anti-angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of the CRCGCLpolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of CRCGCL. Malignant and metastatic conditionswhich can be treated with the polynucleotides and polypeptides, oragonists or antagonists of the invention include, but are not limitedto, malignancies, solid tumors, and cancers described herein andotherwise known in the art (for a review of such disorders, see Fishmanet al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)):

Ocular disorders associated with neovascularization which can be treatedwith the CRCGCL polynucleotides and polypeptides of the presentinvention (including CRCGCL agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978). Additionally, disorders which can betreated with the CRCGCL polynucleotides and polypeptides of the presentinvention (including CRCGCL agonist and/or antagonists) include, but arenot limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated with be treatedwith the CRCGCL polynucleotides and polypeptides of the presentinvention (including CRCGCL agonist and/or antagonists) include, but arenot limited to, solid tumors, blood born tumors such as leukemias, tumormetastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,rheumatoid arthritis, psoriasis, ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, and uvietis, delayed woundhealing, endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated or detected by CRCGCL polynucleotides orpolypeptides, as well as antagonists or agonists of CRCGCL, includecancers (such as follicular lymphomas, carcinomas with p53 mutations,and hormone-dependent tumors, including, but not limited to coloncancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, CRCGCL polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by CRCGCL polynucleotides orpolypeptides, or agonists or antagonists of CRCGCL, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated ordetected by CRCGCL polynucleotides or polypeptides, as well as agonistsor antagonists of CRCGCL, include AIDS; neurodegenerative disorders(such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumoror prior associated disease); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing CRCGCL polynucleotides orpolypeptides, as well as agonists or antagonists of CRCGCL, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. CRCGCLpolynucleotides or polypeptides, as well as agonists or antagonists ofCRCGCL, could be used to promote dermal reestablishment subsequent todermal loss.

CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that CRCGCL polynucleotidesor polypeptides, agonists or antagonists of CRCGCL, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. CRCGCLpolynucleotides or polypeptides, as well as agonists or antagonists ofCRCGCL, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that CRCGCL polynucleotides or polypeptides, as well asagonists or antagonists of CRCGCL, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intesting, and large intestine. CRCGCLpolynucleotides or polypeptides, as well as agonists or antagonists ofCRCGCL, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. CRCGCL polynucleotides or polypeptides, agonists or antagonistsof CRCGCL, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. CRCGCL polynucleotides or polypeptides, as well asagonists or antagonists of CRCGCL, may have a cytoprotective effect onthe small intestine mucosa. CRCGCL polynucleotides or polypeptides, aswell as agonists or antagonists of CRCGCL, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could further be used in full regeneration ofskin in full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. CRCGCLpolynucleotides or polypeptides, as well as agonists or antagonists ofCRCGCL, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. CRCGCL polynucleotides or polypeptides, as well asagonists or antagonists of CRCGCL, could also be used to treat gastricand doudenal ulcers and help heal by scar formation of the mucosallining and regeneration of glandular mucosa and duodenal mucosal liningmore rapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus,CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could be used to promote the resurfacing of themucosal surface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with CRCGCL polynucleotides orpolypeptides, agonists or antagonists of CRCGCL, is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could be used to treat diseases associate withthe under expression of CRCGCL.

Moreover, CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could be used to prevent and heal damage to thelungs due to various pathological states. A growth factor such as CRCGCLpolynucleotides or polypeptides, as well as agonists or antagonists ofCRCGCL, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing CRCGCL polynucleotides or polypeptides, agonists or antagonists ofCRCGCL. Also, CRCGCL polynucleotides or polypeptides, as well asagonists or antagonists of CRCGCL, could be used to stimulate theproliferation of and differentiation of type II pneumocytes, which mayhelp treat or prevent disease such as hyaline membrane diseases, such asinfant respiratory distress syndrome and bronchopulmonary displasia, inpremature infants.

CRCGCL polynucleotides or polypeptides, as well as agonists orantagonists of CRCGCL, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

In addition, CRCGCL polynucleotides or polypeptides, as well as agonistsor antagonists of CRCGCL, could be used treat or prevent the onset ofdiabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, CRCGCL polynucleotidesor polypeptides, as well as agonists or antagonists of CRCGCL, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, CRCGCL polynucleotides orpolypeptides, as well as agonists or antagonists of CRCGCL, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Infectious Disease

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, can be used to treat or detect infectious agents. For example,by increasing the immune response, particularly increasing theproliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by CRCGCL polynucleotides orpolypeptides, or agonists or antagonists of CRCGCL. Examples of viruses,include, but are not limited to the following DNA and RNA viralfamilies: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae(such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus),Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,Rubivirus). Viruses falling within these families can cause a variety ofdiseases or symptoms, including, but not limited to: arthritis,bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis,keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, ChronicActive, Delta), meningitis, opportunistic infections (e.g., AIDS),pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles,Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, can be used to treat or detect any of thesesymptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by CRCGCL polynucleotides orpolypeptides, or agonists or antagonists of CRCGCL, include, but notlimited to, the following Gram-Negative and Gram-positive bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax,Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia,Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), PasteurellaceaInfections (e.g., Actinobacillus, Heamophilus, Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, andStaphylococcal. These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, can be used to treat or detect any of thesesymptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can betreated or detected by CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, include, but not limited to, thefollowing families: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), Malaria, pregnancy complications, andtoxoplasmosis. CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, can be used to treat or detect any of thesesymptoms or diseases.

Preferably, treatment using CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, could either be by administering aneffective amount of CRCGCL polypeptide to the patient, or by removingcells from the patient, supplying the cells with CRCGCL polynucleotide,and returning the engineered cells to the patient (ex vivo therapy).Moreover, the CRCGCL polypeptide or polynucleotide can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, can be used to differentiate, proliferate, and attract cells,leading to the regeneration of tissues. (See, Science 276:59-87 (1997).)The regeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, may increase regeneration of tissues difficult toheal. For example, increased tendon/ligament regeneration would quickenrecovery time after damage. CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, of the present invention could alsobe used prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingCRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, to proliferate and differentiate nerve cells. Diseases thatcould be treated using this method include central and peripheralnervous system diseases, neuropathies, or mechanical and traumaticdisorders (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated using the CRCGCL polynucleotides orpolypeptides, or agonists or antagonists of CRCGCL.

Chemotaxis

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may have chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells,mast cells, eosinophils, epithelial and/or endothelial cells) to aparticular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may increase chemotaxic activity of particular cells. Thesechemotactic molecules can then be used to treat inflammation, infection,hyperproliferative disorders, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat wounds andother trauma to tissues by attracting immune cells to the injuredlocation. As a chemotactic molecule, CRCGCL could also attractfibroblasts, which can be used to treat wounds.

It is also contemplated that CRCGCL polynucleotides or polypeptides, oragonists or antagonists of CRCGCL, may inhibit chemotactic activity.These molecules could also be used to treat disorders. Thus, CRCGCLpolynucleotides or polypeptides, or agonists or antagonists of CRCGCL,could be used as an inhibitor of chemotaxis.

Binding Activity

CRCGCL polypeptides may be used to screen for molecules that bind toCRCGCL or for molecules to which CRCGCL binds. The binding of CRCGCL andthe molecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the CRCGCL or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors),or small molecules.

Preferably, the molecule is closely related to the natural ligand ofCRCGCL, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which CRCGCLbinds, or at least, a fragment of the receptor capable of being bound byCRCGCL (e.g., active site). In either case, the molecule can berationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express CRCGCL, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing CRCGCL(or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either CRCGCL or the molecule.

The assay may simply test binding of a candidate compound to CRCGCL,wherein binding is detected by a label, or in an assay involvingcompetition with a labeled competitor. Further, the assay may testwhether the candidate compound results in a signal generated by bindingto CRCGCL.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining CRCGCL, measuring CRCGCL/molecule activity or binding, andcomparing the CRCGCL/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure CRCGCL level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure CRCGCL level or activity by eitherbinding, directly or indirectly, to CRCGCL or by competing with CRCGCLfor a substrate.

Additionally, the receptor to which CRCGCL binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, expression cloning is employedwherein polyadenylated RNA is prepared from a cell responsive to thepolypeptides, for example, NIH3T3 cells which are known to containmultiple receptors for the FGF family proteins, and SC-3 cells, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to thepolypeptides. Transfected cells which are grown on glass slides areexposed to the polypeptide of the present invention, after they havebeen labelled. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of CRCGCL therebyeffectively generating agonists and antagonists of CRCGCL. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of CRCGCL polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired CRCGCL moleculeby homologous, or site-specific, recombination. In another embodiment,CRCGCL polynucleotides and corresponding polypeptides may be alterred bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of CRCGCL may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are Cytokine Receptor family members. In further preferredembodiments, the heterologous molecule is a growth factor such as, forexample, platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growthfactor (EGF), fibroblast growth factor (FGF), TGF-beta, bonemorphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins Aand B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiationfactors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2,TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active CRCGCL fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the CRCGCL polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the CRCGCL receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the CRCGCL/molecule.Moreover, the assays can discover agents which may inhibit or enhancethe production of CRCGCL from suitably manipulated cells ortissues.Therefore, the invention includes a method of identifyingcompounds which bind to CRCGCL comprising the steps of: (a) incubating acandidate binding compound with CRCGCL; and (b) determining if bindinghas occurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with CRCGCL, (b) assaying a biological activity , and (b)determining if a biological activity of CRCGCL has been altered.

Also, one could identify molecules bind CRCGCL experimentally by usingthe beta-pleated sheet regions disclosed in FIG. 3 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 3/Table 1. Additional embodiments of the invention aredirected to polynucleotides encoding CRCGCL polypeptides which comprise,or alternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 3/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the CRCGCL amino acid sequence ofeach of the beta pleated sheet regions disclosed in FIG. 3/Table 1.Additional embodiments of the invention are directed to CRCGCLpolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions disclosed in FIG.3/Table 1.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 209641 or 209691. In oneembodiment, antisense sequence is generated internally by the organism,in another embodiment, the antisense sequence is separately administered(see, for example, O'Connor, J., Neurochem. 56:560 (1991).Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methodsare based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the CRCGCL antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the CRCGCL antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding CRCGCL, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region (Bemoistand Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc.Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences ofthe metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)),etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a CRCGCLgene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded CRCGCL antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid Generally,the larger the hybridizing nucleic acid, the more base mismatches with aCRCGCL RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of CRCGCL shown in FIGS. 1A-Bcould be used in an antisense approach to inhibit translation ofendogenous CRCGCL mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of CRCGCL mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the CRCGCL coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy CRCGCL mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of CRCGCL (FIGS. 1A-B). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the CRCGCL mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express CRCGCLin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous CRCGCL messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, bums, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat and prevent neuronal damage which occurs incertain neuronal disorders or neuro-degenerative conditions such asAlzheimer's disease, Parkinson's disease, and AIDS-related complex.CRCGCL may have the ability to stimulate chondrocyte growth, therefore,they may be employed to enhance bone and periodontal regeneration andaid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The CRCGCL polypeptide may also be employed for preventing hair loss,since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

The CRCGCL polypeptide may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may also be used to modulate mammalian characteristics, such asbody height, weight, hair color, eye color, skin, percentage of adiposetissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, CRCGCL polynucleotides or polypeptides, or agonists orantagonists of CRCGCL, may be used to modulate mammalian metabolismaffecting catabolism, anabolism, processing, utilization, and storage ofenergy.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may be used to change a mammal's mental state or physical stateby influencing biorhythms, caricadic rhythms, depression (includingdepressive disorders), tendency for violence, tolerance for pain,reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

CRCGCL polynucleotides or polypeptides, or agonists or antagonists ofCRCGCL, may also be used as a food additive or preservative, such as toincrease or decrease storage capabilities, fat content, lipid, protein,carbohydrate, vitamins, minerals, cofactors or other nutritionalcomponents.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of the CRCGCL cDNA Clone from the DepositedSample

The cDNA for CRCGCL is inserted into the EcoRI/XhoI multiple cloningsite of Uni-ZAP XR (Stratagene). Uni-ZAP XR contains an ampicillinresistance gene and may be transformed into E. coli strain DH10B,available from Life Technologies. (See, for instance, Gruber, C. E., etal., Focus 15:59-(1993).)

Two approaches can be used to isolate CRCGCL from the deposited sample.First, the deposited clone is transformed into a suitable host (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents. The transformants are plated on 1.5% agarplates (containing the appropriate selection agent, e.g., ampicillin) toa density of about 150 transformants (colonies) per plate. A singlecolony is then used to generate DNA using nucleic acid isolationtechniques well known to those skilled in the art. (e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), ColdSpring Harbor Laboratory Press.)

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 bounded bythe 5′ NT and the 3′ NT of the clone) are synthesized and used toamplify the CRCGCL cDNA using the deposited cDNA plasmid as a template.The polymerase chain reaction is carried out under routine conditions,for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v)gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94 degree C. for 1 min; annealing at 55 degree C. for 1 min;elongation at 72 degree C. for 1 min) are performed with a Perkin-ElmerCetus automated thermal cycler. The amplified product is analyzed byagarose gel electrophoresis and the DNA band with expected molecularweight is excised and purified. The PCR product is verified to be theselected sequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of the CRCGCL gene which may not be present in thedeposited clone. These methods include but are not limited to, filterprobing, clone enrichment using specific probes, and protocols similaror identical to 5′ and 3′ “RACE” protocols which are well known in theart. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of theCRCGCL gene of interest is used to PCR amplify the 5′ portion of theCRCGCL full-length gene. This amplified product may then be sequencedand used to generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the CRCGCL gene.

Example 2 Isolation of CRCGCL Genomic Clones

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:1, according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of CRCGCL Polypeptides

Tissue distribution of mRNA expression of CRCGCL is determined usingprotocols for Northern blot analysis, described by, among others,Sambrook et al. For example, a CRCGCL probe produced by the methoddescribed in Example 1 is labeled with P³² using the rediprime™ DNAlabeling system (Amersham Life Science), according to manufacturer'sinstructions. After labeling, the probe is purified using CHROMASPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT 1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70 degree C. overnight, and the films developed according to standardprocedures.

Example 4 Chromosomal Mapping of CRCGCL

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:1. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95 degree C.; 1minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32times followed by one 5 minute cycle at 70 degree C. Human, mouse, andhamster DNA is used as template in addition to a somatic cell hybridpanel containing individual chromosomes or chromosome fragments (Bios,Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of CRCGCL

CRCGCL polynucleotide encoding a CRCGCL polypeptide invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

Specifically, to clone the CRCGCL protein in a bacterial vector, the 5′primer has the sequence 5′ GTTAGGCCATGGGAGGAGCAGCAGAAGGA 3′ (SEQ ID NO:14) containing the Nco I restriction site followed a number ofnucleotides of the amino terminal coding sequence of the CRCGCL sequencein SEQ ID NO:1. One of ordinary skill in the art would appreciate, ofcourse, that the point in the protein coding sequence where the 5′primer begins may be varied to amplify a DNA segment encoding anydesired portion of the complete CRCGCL protein shorter or longer thanthe the portion described above. The 3′ primer has the sequence 5′GGTTAAAGATCTCAACGCCACGTAGGAGCGGTC 3′ (SEQ ID NO: 15) containing theBglII restriction site followed by a number nucleotides complementary tothe 3′ end of the coding sequence of the CRCGCL DNA sequence of SEQ IDNO:1.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified CRCGCL protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the CRCGCL protein can be successfullyrefolded while immobilized on the Ni-NTA column. The recommendedconditions are as follows: renature using a linear 6M-1M urea gradientin 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing proteaseinhibitors. The renaturation should be performed over a period of 1.5hours or more. After renaturation the proteins are eluted by theaddition of 250 mM immidazole. Immidazole is removed by a finaldialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200mM NaCl. The purified CRCGCL protein is stored at 4 degree C. or frozenat −80 degree C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a CRCGCL polynucleotide, calledpHE4a. (ATCC Accession Number 209645, deposited Feb. 25, 1998.) Thisvector contains: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,and 6) the lactose operon repressor gene (lacIq). The origin ofreplication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). Thepromoter sequence and operator sequences are made synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system. More preferably, the bacterialexpression vector, pQE60 can also be used to express CRCGCL.

Example 6 Purification of CRCGCL Polypeptide from an Inclusion Body

The following alternative method can be used to purify CRCGCLpolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the CRCGCL polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

The resultant CRCGCL polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Commassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified CRCGCL protein canalso be tested for endotoxin/LPS contamination, and typically the LPScontent is less than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of CRCGCL in a Baculovirus ExpressionSystem

In this example, the plasmnid shuttle vector pA2 is used to insertCRCGCL polynucleotide into a baculovirus to express CRCGCL. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, XbaI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned CRCGCL polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the CRCGCL cDNA sequence contained in the deposited clone,including the AUG initiation codon and any naturally associated leadersequence, is amplified using the PCR protocol described in Example 1. Ifthe naturally occurring signal sequence is used to produce the secretedprotein, the pA2 vector does not need a second signal peptide.Alternatively, the vector can be modified (pA2 GP) to include abaculovirus leader sequence, using the standard methods described inSummers et al., “A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures,” Texas Agricultural Experimental StationBulletin No. 1555 (1987).

More specifically, the cDNA sequence encoding the full length CRCGCLprotein in the deposited clone, including the AUG initiation codon andthe naturally associated leader sequence shown in SEQ ID NO:1, isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the gene. For example, the 5′ primer could have thesequence 5′ CCGGTTAGATCTGCCATCATGGCTTTGGGGCAAGGAGG 3′ (SEQ ID NO: 16)containing the BglII restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.196:947-950 (1987)), followed by a number of nucleotides of the sequenceof the complete CRCGCL protein shown in FIGS. 1A-1B. Alternatively, the5′ primer: 5′ CCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTG 3′ (SEQ ID NO:28),also having a BglII restriction site could also be used. The 3′ primerhas the sequence 5′ CCGGTTTCTAGATCACAAGGCCACGTAGGAGCGGTC 3′ (SEQ ID NO:17) containing the XbaI restriction site followed by a number ofnucleotides complementary to the 3′ noncoding sequence in FIGS. 1A-1B.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transfectedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced CRCGCL protein.

Example 8 Expression of CRCGCL in Mammalian Cells

CRCGCL polypeptide can be expressed in a mammalian cell. A typicalmammalian expression vector contains a promoter element, which mediatesthe initiation of transcription of mRNA, a protein coding sequence, andsignals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2DHFR (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, CRCGCL polypeptide can be expressed in stable cell linescontaining the CRCGCL polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as DHFR, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected CRCGCL gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofCRCGCL. The vectors also contain the 3′ intron, the polyadenylation andtermination signal of the rat preproinsulin gene, and the mouse DHFRgene under control of the SV40 early promoter.

Specifically, the plasmid pC6 or pC4 is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel. Also preferred is the pcDNA3 vector (LifeTechnologies).

If a naturally occurring signal sequence is used to produce a secretedprotein, the vector does not need a second signal peptide.Alternatively, if a naturally occurring signal sequence is not used, thevector can be modified to include a heterologous signal sequence in aneffort to secrete the protein from the cell. (See, e.g., WO 96/34891.)

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The isolated fragment andthe dephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 or pC4using, for instance, restriction enzyme analysis.

For example, a soluble CRCGCL polypeptide, such as amino acids Met 1 to

Lys 231, could also be expressed. A 5′ primer: 5′      CCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTG 3′ (SEQ ID NO:28), having aBgl II restriction site and a 3′ primer: 5′      GGCCGGTCTAGATTATTTGGACAGCTTTGGTTTG 3′ (SEQ ID NO:31)could be used to PCR amino acids Met 1 to Lys 231. The amplified productcould be inserted into a mammalian expression vector, such as pC4 orpC6.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of CRCGCL is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9 Construction of N-terminal and/or C-terminal Deletion Mutants

The following general approach may be used to clone a N-terminal orC-terminal deletion CRCGCL deletion mutant. Generally, twooligonucleotide primers of about 15-25 nucleotides are derived from thedesired 5′ and 3′ positions of a polynucleotide of SEQ ID NO:1. The 5′and 3′ positions of the primers are determined based on the desiredCRCGCL polynucleotide fragment. An initiation and stop codon are addedto the 5′ and 3′ primers respectively, if necessary, to express theCRCGCL polypeptide fragment encoded by the polynucleotide fragment.Preferred CRCGCL polynucleotide fragments are those encoding theN-terminal and C-terminal deletion mutants disclosed above in the“Polynucleotide and Polypeptide Fragments” section of the Specification.

Additional nucleotides containing restriction sites to facilitatecloning of the CRCGCL polynucleotide fragment in a desired vector mayalso be added to the 5′ and 3′ primer sequences. The CRCGCLpolynucleotide fragment is amplified from genomic DNA or from thedeposited cDNA clone using the appropriate PCR oligonucleotide primersand conditions discussed herein or known in the art. The CRCGCLpolypeptide fragments encoded by the CRCGCL polynucleotide fragments ofthe present invention may be expressed and purified in the same generalmanner as the full length polypeptides, although routine modificationsmay be necessary due to the differences in chemical and physicalproperties between a particular fragment and full length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the CRCGCL polypeptide fragment 1-35 to F-276 isamplified and cloned as follows: A 5′ primer is generated comprising arestriction enzyme site followed by an initiation codon in frame withthe polynucleotide sequence encoding the N-terminal portion of thepolypeptide fragment beginning with 1-35. A complementary 3′ primer isgenerated comprising a restriction enzyme site followed by a stop codonin frame with the polynucleotide sequence encoding C-terminal portion ofthe CRCGCL polypeptide fragment ending with F-276.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. TheCRCGCL polynucleotide fragment is inserted into the restrictedexpression vector, preferably in a manner which places the CRCGCLpolypeptide fragment coding region downstream from the promoter. Theligation mixture is transformed into competent E. coli cells usingstandard procedures and as described in the Examples herein. Plasmid DNAis isolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Example 10 Protein Fusions of CRCGCL

CRCGCL polypeptides are preferably fused to other proteins. These fusionproteins can be used for a variety of applications. For example, fusionof CRCGCL polypeptides to His-tag, HA-tag, protein A, IgG domains, andmaltose binding protein facilitates purification. (See Example 5; seealso EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).)Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflifetime in vivo. Nuclear localization signals fused to CRCGCL polypeptidescan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and CRCGCLpolynucleotide, isolated by the PCR protocol described in Example 1, isligated into this BamHI site.

Alternatively, a soluble CRCGCL polypeptide, such as amino acids Met 1to Lys 231, could also be fused to the Fc portion. For Example, a 5′primer: 5′ CCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTG 3′ (SEQ ID NO:28),having a BglII restriction site and a 3 primer: 5′GGCCGGTCTAGATTTGGACAGCTTTGGTTTG 3′ (SEQ ID NO:29) could be used to PCRamino acids Met 1 to Lys 231. The amplified product could be fused to Fcto produce a Fc fusion protein, as set forth above, and ligated to thepC4 vector for mammalian expression or pA2 for baculovirus expression.

In either case, note that the polynucleotide is cloned without a stopcodon, otherwise a fusion protein will not be produced. Moreover, if thenaturally occurring signal sequence is used to produce the secretedprotein, pC4 does not need a second signal peptide. Alternatively, ifthe naturally occurring signal sequence is not used, the vector can bemodified to include a heterologous signal sequence. (See, e.g., WO96/34891.)

The Fc fusions described above could also be inserted into the pA2vector to express in Baculovirus systems, as set forth in Example 7,using techniques known in the art and described herein.

Human IgG Fc region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGG(SEQ ID NO:4)GTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 11 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing CRCGCL is administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of CRCGCL protein is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology.(Kohler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with CRCGCL polypeptide or, more preferably,with a secreted CRCGCL polypeptide-expressing cell. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56 degreeC.), and supplemented with about 10 g/l of nonessential amino acids,about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the CRCGCL polypeptide.

Alternatively, additional antibodies capable of binding to CRCGCLpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the CRCGCLprotein-specific antibody can be blocked byCRCGCL. Such antibodiescomprise anti-idiotypic antibodies to the CRCGCL protein-specificantibody and can be used to immunize an animal to induce formation offurther CRCGCL protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secreted CRCGCLprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

b) Isolation of Antibody Fragments Directed Against CRCGCL from aLibrary of scFvs

Naturally occuring V-genes isolated from human PBLs are constructed intoa large library of antibody fragments which contain reactivities againstCRCGCL to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein in its entirety byreference).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in WO92/01047. To rescue phage displayingantibody fragments, approximately 10⁹ E. coli harbouring the phagemidare used to inoculate 50 ml of 2×TY containing 1% glucose and 100 ug/mlof ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU,2×10⁸ TU of delta gene 3 helper (M13 delta gene III, see WO92/01047) areadded and the culture incubated at 37 degree C. for 45 minutes withoutshaking and then at 37 degree C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of of 2×TY containing 100 ug/ml ampicillin and50 ug/ml kanamycin and grown overnight. Phage are prepared as describedin WO092/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harbouring a pUC19 derivative supplying the wild typegene III protein during phage morphogenesis. The culture is incubatedfor 1 hour at 37 degree C. without shaking and then for a further hourat 37 degree C. with shaking. Cells are spun down (IEC-Centra 8, 4000revs/min for 10 min), resuspended in 300 ml 2×TY broth containing 100 ugampicillin/ml and 25 ug kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 um filter (MinisartNML; Sartorius) to give a final concentration of approximately 10¹³transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 ug/ml or 10 ug/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37 degree C. and then washed 3 times in PBS. Approximately 10¹³ TU ofphage is applied to the tube and incubated for 30 minutes at roomtemperature tumbling on an over and under turntable and then left tostand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1%Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100mM triethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37 degreeC. The E. coli are then plated on TYE plates containing 1% glucose and100 ug/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (seee.g., WO92/01047) and then by sequencing.

Example 12 Production of CRCGCL Protein for High-throughput ScreeningAssays

The following protocol produces a supernatant containing CRCGCLpolypeptide to be tested. This supernatant can then be used in theScreening Assays described in Examples 14-21.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503FBiowhittaker)/1×Penstrep(17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples8-10, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37 degree C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1×penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2 (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄-H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamineand 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1 L DMEM fora 10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degree C. for 45 or 72 hours depending on the mediaused: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 14-21.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the CRCGCL polypeptide directly (e.g., as a secretedprotein) or by CRCGCL inducing expression of other proteins, which arethen secreted into the supernatant. Thus, the invention further providesa method of identifying the protein in the supernatant characterized byan activity in a particular assay.

Example 13 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in Table2 below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proxial region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:5)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table 2 below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified.

There is preliminary data that CRCGCL interacts with Jak1.

TABLE 2 JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISRE IFNfamily IFN-a/B + + − − 1,2,3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP)Il-10 + ? ? − 1,3 gp130 family IL-6 (Pleiotrohic) + + + ? 1,3 GAS(IRF1 > Lys6 > IFP) Il-11 (Pleiotrohic) ? + ? ? 1,3 OnM (Pleiotrohic)? + + ? 1,3 LIF (Pleiotrohic) ? + + ? 1,3 CNTF (Pleiotrohic) −/+ + + ?1,3 G-CSF (Pleiotrohic) ? + ? ? 1,3 IL-12 (Pleiotrohic) + − + + 1,3 g-Cfamily IL-2 (lymphocytes) − + − + 1,3,5 GAS IL-4 (lymph/myeloid) − + − +6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9(lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? +? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6)IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1,3,5 EPO ? − + − 5 GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3GAS (IRF1) PDGF ? + + − 1,3 CSF-1 ? + + − 1,3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 14-15, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris:

5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC (SEQ ID NO:6)GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and isflanked with a HindIII site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ IDNO:7).

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/HindIII and subcloned into BLSK2−.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:

5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA (SEQ ID NO:8)TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 14-15.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NFK-B and EGR promoter sequences aredescribed in Examples 16 and 17. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 14 High-throughput Screening Assay for T-cell Activity

The following protocol is used to assess T-cell activity of CRCGCL bydetermining whether CRCGCL supernatant proliferates and/ordifferentiates T-cells. T-cell activity is assessed using theGAS/SEAP/Neo construct produced in Example 13. Thus, factors thatincrease SEAP activity indicate the ability to activate the Jaks-STATSsignal transduction pathway. The T-cell used in this assay is JurkatT-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCCAccession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582)cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing CRCGCL polypeptides or CRCGCL inducedpolypeptides as produced by the protocol described in Example 12.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48-72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degree C.until SEAP assays are performed according to Example 18. The platescontaining the remaining treated cells are placed at 4 degree C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

Example 15 High-throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of CRCGCL bydetermining whether CRCGCL proliferates and/or differentiates myeloidcells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 13. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The myeloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 13, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e⁷U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degree C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 12. Incubate at 37 degree C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 18.

Example 16 High-throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed by CRCGCL.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC 12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells by CRCGCL can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers:

5′GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:9)5′GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO:10)

Using the GAS:SEAP/Neo vector produced in Example 13, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 12. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 12, 37 degree C. for 48 to 72 hr. As a positive control, agrowth factor known to activate PC12 cells through EGR can be used, suchas 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold inductionof SEAP is typically seen in the positive control wells. SEAP assay thesupernatant according to Example 18.

Example 17 High-throughput Screening Assay for T-cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a widevariety of agents including the inflammatory cytokines IL-1 and TNF,CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure toLPS or thrombin, and by expression of certain viral gene products. As atranscription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm withI-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylatedand degraded, causing NF-KB to shuttle to the nucleus, therebyactivating transcription of target genes. Target genes activated byNF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KB promoter element are used toscreen the supernatants produced in Example 12. Activators or inhibitorsof NF-KB would be useful in treating diseases. For example, inhibitorsof NF-KB could be used to treat those diseases related to the acute orchronic activation of NF-KB, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR basedstrategy is employed. The upstream primer contains four tandem copies ofthe NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:11), 18 bp of sequencecomplementary to the 5′ end of the SV40 early promoter sequence, and isflanked with an XhoI site:

5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC (SEQ ID NO:12)TTTCCATCCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a HindIII site:

-   5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:7)

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and HindIII and subcloned into BLSK2−.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence:

5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC (SEQ ID NO:13)ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT:3′

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAPcassette is removed from the above NF-KB/SEAP vector using restrictionenzymes SalI and NotI, and inserted into a vector containing neomycinresistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted intopGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 withSalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells arecreated and maintained according to the protocol described in Example14. Similarly, the method for assaying supernatants with these stableJurkat T-cells is also described in Example 14. As a positive control,exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11,with a 5-10 fold activation typically observed.

Example 18 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 14-17, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5×Dilution Buffer and dispense 15 ul of2.5×dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see Table 3 below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint results. An increase in chemiluminescence indicates reporteractivity.

TABLE 3 Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml)CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 1690 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 623 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 1859.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 21510.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 24512.25 48 250 12.5 49 255 12.75 50 260 13

Example 19 High-throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule,fluo-3, used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acid DMSO. Toload the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is added to eachwell. The plate is incubated at 37 degree C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-3 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension.

The tube is then placed in a 37 degree C. water bath for 30-60 min. Thecells are washed twice with HBSS, resuspended to 1×10⁶ cells/ml, anddispensed into a microplate, 100 ul/well. The plate is centrifuged at1000 rpm for 5 min. The plate is then washed once in Denley CellWashwith 200 ul, followed by an aspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-3. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by the a molecule, either CRCGCL or a molecule induced by CRCGCL,which has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 20 High-throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies.

In addition there are a large family of RPTKs for which thecorresponding ligand is unknown. Ligands for RPTKs include mainlysecreted small proteins, but also membrane-bound and extracellularmatrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether CRCGCL or a moleculeinduced by CRCGCL is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 12, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of proteaseinhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4° C. The plate is then placed in a vacuum transfermanifold and the extract filtered through the 0.45 mm membrane bottomsof each well using house vacuum. Extracts are collected in a 96-wellcatch/assay plate in the bottom of the vacuum manifold and immediatelyplaced on ice. To obtain extracts clarified by centrifugation, thecontent of each well, after detergent solubilization for 5 minutes, isremoved and centrifuged for 15 minutes at 4 degree C at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5×Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degree C. for 20 min. This allows the streptavadin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml))to each well and incubate at 37 degree C. for one hour. Wash the well asabove.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 21 High-throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 20, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 12 for 5-20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by CRCGCL or a molecule inducedby CRCGCL.

Example 22 Method of Determining Alterations in the CRCGCL Gene

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95 degree C. for 30 seconds; 60-120seconds at 52-58 degree C.; and 60-120 seconds at 70 degree C., usingbuffer solutions described in Sidransky, D., et al., Science 252:706(1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofCRCGCL is also determined and genomic PCR products analyzed to confirmthe results. PCR products harboring suspected mutations in CRCGCL isthen cloned and sequenced to validate the results of the directsequencing.

PCR products of CRCGCL are cloned into T-tailed vectors as described inHolton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991)and sequenced with T7 polymerase (United States Biochemical). Affectedindividuals are identified by mutations in CRCGCL not present inunaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in the CRCGCL gene. Genomic clones isolated according toExample 2 are nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH performed as described inJohnson, Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridizationwith the labeled probe is carried out using a vast excess of human cot-1DNA for specific hybridization to the CRCGCL genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of CRCGCL (hybridized by the probe)are identified as insertions, deletions, and translocations. TheseCRCGCL alterations are used as a diagnostic marker for an associateddisease.

Example 23 Method of Detecting Abnormal Levels of CRCGCL in a BiologicalSample

CRCGCL polypeptides can be detected in a biological sample, and if anincreased or decreased level of CRCGCL is detected, this polypeptide isa marker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect CRCGCL in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to CRCGCL, at a final concentration of0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal andare produced by the method described in Example 11. The wells areblocked so that non-specific binding of CRCGCL to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining CRCGCL. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withdeionized or distilled water to remove unbounded CRCGCL.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot CRCGCL polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the CRCGCL in the sample using the standard curve.

Example 24 Formulating a Polypeptide

The CRCGCL composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the CRCGCL polypeptide alone), the site of delivery, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The “effective amount” for purposesherein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofCRCGCL administered parenterally per dose will be in the range of about1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, CRCGCL is typically administered at a dose rate of about 1ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

Pharmaceutical compositions containing CRCGCL are administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

CRCGCL is also suitably administered by sustained-release systems.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed.Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped CRCGCL polypeptides.Liposomes containing the CRCGCL are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, CRCGCL is formulatedgenerally by mixing it at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting CRCGCL uniformlyand intimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

CRCGCL is typically formulated in such vehicles at a concentration ofabout 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3to 8. It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.

CRCGCL used for therapeutic administration can be sterile. Sterility isreadily accomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

CRCGCL polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous CRCGCL polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized CRCGCL polypeptide usingbacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, CRCGCLmay be employed in conjunction with other therapeutic compounds.

The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 25 Method of Treating Decreased Levels of CRCGCL

The present invention relates to a method for treating an individual inneed of a decreased level of CRCGCL activity in the body comprising,administering to such an individual a composition comprising atherapeutically effective amount of CRCGCL antagonist. Preferredantagonists for use in the present invention are CRCGCL-specificantibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of CRCGCL in an individual canbe treated by administering CRCGCL, preferably in the secreted form.Thus, the invention also provides a method of treatment of an individualin need of an increased level of CRCGCL polypeptide comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of CRCGCL to increase the activity level of CRCGCLin such an individual.

For example, a patient with decreased levels of CRCGCL polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 24.

Example 26 Method of Treating Increased Levels of CRCGCL

The present invention also relates to a method for treating anindividual in need of an increased level of CRCGCL activity in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of CRCGCL or an agonist thereof.

Antisense technology is used to inhibit production of CRCGCL. Thistechnology is one example of a method of decreasing levels of CRCGCLpolypeptide, preferably a secreted form, due to a variety of etiologies,such as cancer.

For example, a patient diagnosed with abnormally increased levels ofCRCGCL is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if the treatment was well tolerated. Theformulation of the antisense polynucleotide is provided in Example 24.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing CRCGCL polypeptides, onto a patient. Generally, fibroblastsare obtained from a subject by skin biopsy. The resulting tissue isplaced in tissue-culture medium and separated into small pieces. Smallchunks of the tissue are placed on a wet surface of a tissue cultureflask, approximately ten pieces are placed in each flask. The flask isturned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and thechunks of tissue remain fixed to the bottom of the flask and fresh media(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) isadded. The flasks are then incubated at 37 degree C. for approximatelyone week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding CRCGCL can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB 101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted CRCGCL.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the CRCGCL gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the CRCGCL gene(the packagingcells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether CRCGCLprotein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 28 Gene Therapy Using Endogenous CRCGCL Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous CRCGCL sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous CRCGCL, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of CRCGCL so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousCRCGCL sequence. This results in the expression of CRCGCL in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the CRCGCL locus, plasmid pUC18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′end. Two CRCGCL non-coding sequences are amplified viaPCR: one CRCGCL non-coding sequence (CRCGCL fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′end; theother CRCGCL non-coding sequence (CRCGCL fragment 2) is amplified with aBamHI site at the 5′end and a HindIII site at the 3′end. The CMVpromoter and CRCGCL fragments are digested with the appropriate enzymes(CMV promoter—XbaI and BamHI; CRCGCL fragment 1—XbaI; CRCGCL fragment2—BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μ/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) CRCGCL sequences into an animal to increase ordecrease the expression of the CRCGCL polypeptide. The CRCGCLpolynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the CRCGCL polypeptideby the target tissue. Such gene therapy and delivery techniques andmethods are known in the art, see, for example, WO90/11092, WO98/11779;U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al. (1997)Cardiovasc. Res. 35(3):470-479, Chao J et al. (1997) Pharmacol. Res.35(6):517-522, Wolff J. A. (1997) Neuromuscul. Disord. 7(5):314-318,Schwartz B. et al. (1996) Gene Ther. 3(5):405-411, Tsurumi Y. et al.(1996) Circulation 94(12):3281-3290 (incorporated herein by reference).

The CRCGCL polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The CRCGCL polynucleotideconstructs can be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the CRCGCL polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.(1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995)Biol. Cell 85(1):1-7) which can be prepared by methods well known tothose skilled in the art.

The CRCGCL polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

The CRCGCL polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked CRCGCL polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked CRCGCLpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected CRCGCL polynucleotide in muscle invivo is determined as follows. Suitable CRCGCL template DNA forproduction of mRNA coding for CRCGCL polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The CRCGCL template DNA is injected in 0.1 ml of carrier ina 1 cc syringe through a 27 gauge needle over one minute, approximately0.5 cm from the distal insertion site of the muscle into the knee andabout 0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for CRCGCL protein expression. A time course for CRCGCL proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of CRCGCLDNA in muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using CRCGCL naked DNA.

Example 30 CRCGCL Transgenic Animals

The CRCGCL polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate transgenic animals. In a specific embodiment, techniquesdescribed herein or otherwise known in the art, are used to expresspolypeptides of the invention in humans, as part of a gene therapyprotocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

Any of the CRCGCL polypeptides disclose throughout this application canbe used to generate transgenic animals. For example, DNA encoding aminoacids M1-K231 of SEQ ID NO:2 can be inserted into a vector containing apromoter, such as the actin promoter, which will ubiquitously expressthe inserted fragment. Primers that can be used to generate suchfragments include a 5′ primer containing a BglII restriction site shownin bold:

CCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTGCCGGTTAGATCTGC (SEQ ID NO:32)CATCATGGGGCGGCTGGTTCTGCCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTGCCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTGand a 3′ primer, containing a Xba restriction site shown in bold:GGCCGGTCTAGATTATTTGGACAGCTTTGGTTTG (SEQ ID NO: 31). This construct willexpress the predicted soluble domain of CRCGCL under the control of theactin promoter for ubiquitous expression. The region of CRCGCL includedin this construct extends from M1-K231 of SEQ ID NO:2.

Similarly, the DNA encoding the full length CRCGCL protein can also beinserted into a vector.

Alternatively, polynucleotides of the invention can be inserted in avector which controls tissue specific expression through a tissuespecific promoter. For example, a construct having a transferrinpromoter would express the CRCGCL polypeptide in the liver of transgenicanimals. Therefore, DNA encoding amino acids M1-K231 of SEQ ID NO:2 canbe amplified using a 5′ primer, having a BglII restriction site shown inbold: CCGGTTAGATCTGCCATCATGGGGCGGCTGGTTCTG (SEQ ID NO: 28), and a 3′primer, containing a Xba restriction site shown in bold:GGCCGGTCTAGATTATTTGGACAGCTTTGGTTTG (SEQ ID NO: 31).

Similarly, the DNA encoding the full length CRCGCL protein can also beinserted into a vector for tissue specific expression.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of CRCGCL polypeptides, studying conditions and/or disordersassociated with aberrant CRCGCL expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 31 CRCGCL Knock-out Animals

Endogenous CRCGCL gene expression can also be reduced by inactivating or“knocking out” the CRCGCL gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe CRCGCL polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of CRCGCL polypeptides, studying conditions and/or disordersassociated with aberrant CRCGCL expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.For example, a knock-out mouse can be made using the sequences disclosedas AA008694 and W98372, herein incorporated by reference in theirentirety.

Example 32 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10 , IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified CRCGCL protein, or truncated forms thereof, isassessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of CRCGCL protein on purified humantonsillar B cells, measured qualitatively over the dose range from 0.1to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10ug/mil streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of CRCGCL protein, or truncated forms thereof. Micereceive this treatment for 4 consecutive days, at which time they aresacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and CRCGCL protein-treatedspleens identify the results of the activity of CRCGCL protein on spleencells, such as the diffusion of peri-arterial lymphatic sheaths, and/orsignificant increases in the nucleated cellularity of the red pulpregions, which may indicate the activation of the differentiation andproliferation of B-cell populations. Immunohistochemical studies using aB cell marker, anti-CD45R(B220), are used to determine whether anyphysiological changes to splenic cells, such as splenic disorganization,are due to increased B-cell representation within loosely defined B-cellzones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from CRCGCL protein-treated miceis used to indicate whether CRCGCL protein specifically increases theproportion of ThB+, CD45R(B220)dull B cells over that which is observedin control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andCRCGCL protein-treated mice.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 33 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B 33.1) overnight at 4° C.(1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three timeswith PBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of CRCGCL protein (total volume 200 μl). Relevantprotein buffer and medium alone are controls. After 48 hr. culture at37° C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatantis removed and stored −20° C. for measurement of IL-2 (or othercytokines) if effect on proliferation is observed. Wells aresupplemented with 100 μl of medium containing 0.5 μCi of ³H-thymidineand cultured at 37° C. for 18-24 hr. Wells are harvested andincorporation of ³H-thymidine used as a measure of proliferation.Anti-CD3 alone is the positive control for proliferation. IL-2 (100U/ml) is also used as a control which enhances proliferation. Controlantibody which does not induce proliferation of T cells is used as thenegative controls for the effects of CRCGCL proteins.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 34 Effect of CRCGCL on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCYRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of CRCGCL or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofCRCGCL for 24 hours. LPS (100 ng/ml) is added to the cell culture aspositive control. Supernatants from the cell cultures are then collectedand analyzed for IL-12 content using commercial ELISA kit (e.g, R & DSystems (Minneapolis, Minn.)). The standard protocols provided with thekits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of CRCGCLor LPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. CRCGCL, agonists, or antagonists of CRCGCL can be screenedusing the three assays described below. For each of these assays,Peripheral blood mononuclear cells (PBMC) are purified from single donorleukopacks (American Red Cross, Baltimore, Md.) by centrifugationthrough a Histopaque gradient (Sigma). Monocytes are isolated from PBMCby counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubated atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of CRCGCL and under the same conditions,but in the absence of CRCGCL. For IL-12 production, the cells are primedovernight with IFN (100 U/ml) in presence of CRCGCL. LPS (10 ng/ml) isthen added. Conditioned media are collected after 24 h and kept frozenuntil use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is thenperformed using a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of CRCGCL are added to the wells ina total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamineand antibiotics). After 3 days incubation, the plates are centrifugedand the medium is removed from the wells. To the macrophage monolayers,0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassiumphosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/mlof HRPO) is added, together with the stimulant (200 nM PMA). The platesare incubated at 37° C. for 2 hours and the reaction is stopped byadding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. Tocalculate the amount of H₂O₂ produced by the macrophages, a standardcurve of a H₂O₂ solution of known molarity is performed for eachexperiment.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 35 CRCGCL Biological Effects

Astrocyte and Neuronal Assays

Recombinant CRCGCL, expressed in Escherichia coli and purified asdescribed above, can be tested for activity in promoting the survival,neurite outgrowth, or phenotypic differentiation of cortical neuronalcells and for inducing the proliferation of glial fibrillary acidicprotein immunopositive cells, astrocytes. The selection of corticalcells for the bioassay is based on the prevalent expression of FGF-1 andFGF-2 in cortical structures and on the previously reported enhancementof cortical neuronal survival resulting from FGF-2 treatment. Athymidine incorporation assay, for example, can be used to elucidateCRCGCL's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke, P. et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of CRCGCL toinduce neurite outgrowth can be compared to the response achieved withFGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and endothelial cell assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE₂ assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or CRCGCL with or without IL-1α for 24 hours. The supernatants arecollected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.).For the IL-6 assays, the human lung fibroblasts are cultured at 5,000cells/well in a 96-well plate for one day. After a medium change to 0.1%BSA basal medium, the cells are incubated with FGF-2 or CRCGCL with orwithout IL-1α for 24 hours. The supernatants are collected and assayedfor IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or CRCGCL for 3 days inbasal medium before the addition of Alamar Blue to assess effects ongrowth of the fibroblasts. FGF-2 should show a stimulation at 10-2500ng/ml which can be used to compare stimulation with CRCGCL.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, CRCGCL can be evaluated to determinewhether it has an action similar to that of FGF-2 in enhancingdopaminergic neuronal survival in vitro and it can also be tested invivo for protection of dopaminergic neurons in the striatum from thedamage associated with MPTP treatment. The potential effect of CRCGCL isfirst examined in vitro in a dopaminergic neuronal cell cultureparadigm. The cultures are prepared by dissecting the midbrain floorplate from gestation day 14 Wistar rat embryos. The tissue isdissociated with trypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells are maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (N1). The cultures are fixed with paraformaldehyde after 8days in vitro and are processed for tyrosine hydroxylase, a specificmarker for dopminergic neurons, immunohistochemical staining.Dissociated cell cultures are prepared from embryonic rats. The culturemedium is changed every third day and the factors are also added at thattime.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if CRCGCL acts to prolong the survival of dopaminergicneurons, it would suggest that CRCGCL may be involved in Parkinson'sDisease.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 36 The Effect of CRCGCL on the Growth of Vascular EndothelialCells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. CRCGCLprotein of SEQ ID NO. 2, and positive controls, such as VEGF and basicFGF (bFGF) are added, at varying concentrations. On days 4 and 6, themedium is replaced. On day 8, cell number is determined with a CoulterCounter.

An increase in the number of HUVEC cells indicates that CRCGCL mayproliferate vascular endothelial cells.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 37 Stimulatory Effect of CRCGCL on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or CRCGCL in 0.5% FBS) with or withoutHeparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMSmixture (1:0.05) are added per well and allowed to incubate for 1 hourat 37° C. before measuring the absorbance at 490 nm in an ELISA platereader. Background absorbance from control wells (some media, no cells)is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518(1994).

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 38 Inhibition of PDGF-induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4° C. for 2 h after being exposed todenaturing solution and then incubated with the streptavidin-peroxidaseand diaminobenzidine. After counterstaining with hematoxylin, the cellsare mounted for microscopic examination, and the BrdUrd-positive cellsare counted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 39 Stimulation of Endothelial Migration

This example will be used to explore the possibility that CRCGCL maystimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 40 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, CRCGCL activity canbe assayed by determining nitric oxide production by endothelial cellsin response to CRCGCL.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andCRCGCL. Nitric oxide in the medium is determined by use of the Griessreagent to measure total nitrite after reduction of nitric oxide-derivednitrate by nitrate reductase. The effect of CRCGCL on nitric oxiderelease is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 41 Effect of CRCGCL on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or CRCGCL (0.1 to 100 ng/ml) and thecells are cultured for an additional 48 hr. The numbers and lengths ofthe capillary-like chords are quantitated through use of the BoeckelerVIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 42 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of CRCGCL to stimulate angiogenesis in CAMcan be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 43 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of CRCGCL measures the ability of an existingcapillary network to form new vessels in an implanted capsule of murineextracellular matrix material (Matrigel). The protein is mixed with theliquid Matrigel at 4 degree C. and the mixture is then injectedsubcutaneously in mice where it solidifies. After 7 days, the solid“plug” of Matrigel is removed and examined for the presence of new bloodvessels. Matrigel is purchased from Becton DickinsonLabware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with CRCGCL at 150 ng/ml at 4 degree C. and drawn intocold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old areinjected with the mixture of Matrigel and experimental protein at 2sites at the midventral aspect of the abdomen (0.5 ml/site). After 7days, the mice are sacrificed by cervical dislocation, the Matrigelplugs are removed and cleaned (i.e., all clinging membranes and fibroustissue is removed). Replicate whole plugs are fixed in neutral buffered10% formaldehyde, embedded in paraffin and used to produce sections forhistological examination after staining with Masson's Trichrome. Crosssections from 3 different regions of each plug are processed. Selectedsections are stained for the presence of vWF. The positive control forthis assay is bovine basic FGF (150 ng/ml). Matrigel alone is used todetermine basal levels of angiogenesis.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of CRCGCL on ischemia, a rabbit hindlimbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita, S. et al., Am J. Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al. Am J. Pathol 147:1649-1660 (1995)). An interval of10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked CRCGCLexpression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al. HumGene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin. Invest. 90:936-944 (1992)). When CRCGCL is used in the treatment, a single bolus of500 mg CRCGCL protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 min. through an infusioncatheter. On day 30, various parameters are measured in these rabbits:(a) BP ratio—The blood pressure ratio of systolic pressure of theischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 45 Effect of CRCGCL on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of CRCGCL to affect the blood pressure inspontaneously hypertensive rats (SHR) is examined. Increasing doses (0,10, 30, 100, 300, and 900 mg/kg) of the CRCGCL are administered to 13-14week old spontaneously hypertensive rats (SHR). Data are expressed asthe mean+/−SEM. Statistical analysis are performed with a paired t-testand statistical significance is defined as p<0.05 vs. the response tobuffer alone.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 46 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. CRCGCL expression, during the skin ischemia, is studied usingin situ hybridization.

The study in this model is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

c) Topical treatment with CRCGCL of the excisional wounds (day 0, 1, 2,3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100mg.

d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 47 Peripheral Arterial Disease Model

Angiogenic therapy using CRCGCL is a novel therapeutic strategy toobtain restoration of blood flow around the ischemia in case ofperipheral arterial diseases. The experimental protocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) CRCGCL protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of CRCGCL expression andhistology. Biopsy is also performed on the other side of normal muscleof the contralateral hindlimb.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 48 Ischemic Myocardial Disease Model

CRCGCL is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of CRCGCL expression isinvestigated in situ. The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) CRCGCL protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-4 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 49 Rat Corneal Wound Healing Model

This animal model shows the effect of CRCGCL on neovascularization. Theexperimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

b) Inserting a spatula below the lip of the incision facing the outercorner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of CRCGCL, within thepocket.

e) CRCGCL treatment can also be applied topically to the corneal woundsin a dosage range of 20 mg-500 mg (daily treatment for five days).

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 50 Diabetic Mouse and Glucocorticoid-impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that CRCGCL accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et a., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

CRCGCL is administered using at a range different doses of CRCGCL, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) CRCGCL.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:

 [Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with CRCGCL. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (197 8)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that CRCGCL can accelerate the healing process, theeffects of multiple topical applications of CRCGCL on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

CRCGCL is administered using at a range different doses of CRCGCL, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) CRCGCL treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith CRCGCL. A calibrated lens micrometer is used by a blinded observerto determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 51 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofCRCGCL in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7-10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80 EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

Example 52 Suppression of TNF Alpha-induced Adhesion Molecule Expressionby CRCGCL

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of CRCGCL to mediate a suppression of TNF-a induced CAMexpression can be examined. A modified ELISA assay which uses ECs as asolid phase absorbent is employed to measure the amount of CAMexpression on TNF-a treated ECs when co-stimulated with a member of theFGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca, Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca, Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed ×3 with PBS(+Ca, Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10°)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity in CRCGCL protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of CRCGCL polynucleotides (e.g., genetherapy), agonists, and/or antagonists of CRCGCL.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Moreover, the sequence listing from U.S. application Ser. No.60/078,563, filed Mar. 19, 1998, Ser. No. 60/086,505, filed May 22,1998, and Ser. No. 09/263,626, filed Mar. 5, 1999, are hereinincorporated by reference.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence comprisingresidues +1 to +371 of SEQ ID NO:2; (b) an amino acid sequencecomprising residues +2 to +371 of SEQ ID NO:2; and (c) an amino acidsequence comprising residues +23 to +371 of SEQ ID NO:2.
 2. The isolatedpolypeptide of claim 1 which comprises amino acid sequence (a).
 3. Theisolated polypeptide of claim 1 which comprises amino acid sequence (b).4. The isolated polypeptide of claim 1 which comprises amino acidsequence (c).
 5. The isolated polypeptide of claim 1 wherein said aminoacid sequence further comprises a heterologous polypeptide sequence. 6.The isolated polypeptide of claim 5 wherein said heterologouspolypeptide sequence is that of the Fc domain of immunoglobulin.
 7. Acomposition comprising the isolated polypeptide of claim 1 and atpharmaceutically acceptable carrier.
 8. An isolated heterodimercomprising the isolated polypeptide of claim
 1. 9. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence of the full length polypeptideencoded by the cDNA in ATCC Deposit No. 209691 or 209641; (b) an aminoacid sequence of the full length polypeptide excluding the N-terminalmethionine residue, encoded by the cDNA in ATCC Deposit No. 209691 or209641; and (c) an amino acid sequence of the mature polypeptide encodedby the cDNA in ATCC Deposit No. 209691 or
 209641. 10. The isolatedpolypeptide of claim 9 which comprises amino acid sequence (a).
 11. Theisolated polypeptide of claim 9 which comprises amino acid sequence (b).12. The isolated polypeptide of claim 9 which comprises amino acidsequence (c).
 13. The isolated polypeptide of claim 9 wherein said aminoacid sequence further comprises a heterologous polypeptide sequence. 14.The isolated polypeptide of claim 13 wherein said heterologouspolypeptide sequence is that of the Fc domain of immunoglobulin.
 15. Acomposition comprising the isolated polypeptide of claim 9 and apharmaceutically acceptable carrier.
 16. An isolated heterodimercomprising the isolated polypeptide of claim
 9. 17. An isolatedpolypeptide consisting of amino acid residues +1 to +231 of SEQ ID NO:2.18. An isolated polypeptide consisting of amino acid residues selectedfrom the group consisting of: (a) amino acid residues +23 to +231 or SEQID NO:2; (b) amino acid residues +23 to +225 of SEQ ID NO:2; and (c)amino acid residues +226 to +260 of SEQ ID NO:2.
 19. An isolatedpolypeptide consisting of an amino acid sequence selected from the groupconsisting of: (a) amino acid residues +22 to +29 of SEQ ID NO:2; (b)amino acid residues +48 to +56 of SEQ ID NO:2; (c) amino acid residues+62 to +73 of SEQ ID NO:2; (d) amino acid residues +78 to +85 of SEQ IDNO:2; (e) amino acid residues +88 to +95 of SEQ ID NO:2; (f) amino acidresidues +99 to +105 of SEQ ID NO:2; (g) amino acid residues +118 to+126 of SEQ ID NO:2; (h) amino acid residues +139 to +146 of SEQ IDNO:2; (i) amino acid residues +151 to +169 of SEQ ID NO:2; (j) aminoacid residues +188 to +206 of SEQ ID NO:2; (k) amino acid residues +208to +231 of SEQ ID NO:2; (l) amino acid residues +264 to +271 of SEQ IDNO:2; (m) amino acid residue; +286 to +293 of SEQ ID NO:2; (n) aminoacid residues +300 to +313 of SEQ ID NO:2; (o) amino acid residues +317to +342 of SEQ ID NO:2; (p) amino acid residues +347 to +353 of SEQ IDNO:2; and (q) amino acid residues +363 to +369 of SEQ ID NO:2, whereinthe polypeptide consisting of said amino acid sequence as fused to aheterologous polypeptide.
 20. An isolated polypeptide consisting of atleast 30 contiguous amino acid residues of SEQ ID NO:2.
 21. The isolatedpolypeptide of claim 20, consisting of at least 50 contiguous amino acidresidues of SEQ ID NO:2.
 22. The isolated polypeptide of claim 20wherein said polypeptide inhibits the differentiation and/orproliferation of immune cells.
 23. An isolated polypeptide consisting ofat least 30 contiguous amino acid residues encoded by the cDNA in ATCCDeposit No. 209691 or
 209641. 24. The isolated polypeptide of claim 23,consisting of at least 50 contiguous amino acid residues encoded by thecDNA in ATCC Deposit No. 209691 to
 209641. 25. The isolated polypeptideof claim 23 wherein said polypeptide inhibits the differentiation and/orproliferation of immune cells.
 26. An isolated polypeptide comprising afirst amino acid sequence 90% or more identical to a second amino acidsequence selected from the group consisting of: (a) amino acids +1 to+371 of SEQ ID NO:2; (b) amino acids +2 to +371 of SEQ ID NO:2; and (c)amino acids +23 to +371 of SEQ ID NO:2; wherein the isolated polypeptidecomprising said first amino acid sequence stimulates immune cellproliferation.
 27. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 90% or more identical to said second aminoacid sequence (a).
 28. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 90% or more identical to said second aminoacid sequence (b).
 29. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 90% or more identical to said second aminoacid sequence (c).
 30. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 95% or more identical to said second aminoacid sequence (a).
 31. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 95% or more identical to said second aminoacid sequence (b).
 32. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence is 95% or more identical to said second aminoacid sequence (c).
 33. The isolated polypeptide of claim 26 wherein saidfirst amino acid sequence further comprises a heterologous polypeptidesequence.
 34. The isolated polypeptide of claim 33 wherein saidheterologous polypeptide is the Fc domain of immunoglobulin.
 35. Acomposition comprising the isolated polypeptide of claim 26 and apharmaceutically acceptable carrier.
 36. An isolated polypeptidecomprising a first amino acid sequence 90% or more identical to a secondamino acid sequence selected from the group consisting of: (a) an aminoacid sequence of the full length polypeptide encoded by the cDNA in ATCCDeposit No. 209691 or 209641; (b) an amino acid sequence of the fulllength polypeptide, excluding the N-terminal methionine residue, encodedby the cDNA in ATCC Deposit No. 200691 or 209641; and (c) am amino acidsequence of the mature polypeptide encoded by the cDNA in ATCC DepositNo. 209691 or 209641; wherein the polypeptide comprising said firstamino acid sequence stimulates immune cell proliferation.
 37. Theisolated polypeptide of claim 36 wherein said first amino acid sequenceis 90% or more identical to said second amino acid sequence (a).
 38. Theisolated polypeptide of claim 36 wherein said first amino acid sequenceis 90% or more identical to said second amino acid sequence (b).
 39. Theisolated polypeptide of claim 36 wherein said first amino acid sequenceis 90% or more identical to said second amino acid sequence (c).
 40. Theisolated polypeptide at claim 36 wherein said first amino acid sequenceis 95% or more identical to said second amino acid sequence (a).
 41. Theisolated polypeptide of claim 36 wherein said first amino acid sequenceis 95% or more identical to said second amino acid sequence (b).
 42. Theisolated polypeptide of claim 36 wherein said first amino acid sequenceis 95% or more identical to said second amino acid sequence (c).
 43. Theisolated polypeptide claim 36 wherein said polypeptide further comprisesa heterologous polypeptide sequence.
 44. The isolated polypeptide ofclaim 43 wherein said heterologous polypeptide is the Fc domain ofimmunoglobulin.
 45. A composition comprising the isolated polypeptide ofclaim 36 and a pharmaceutically acceptable carrier.
 46. An isolatedpolypeptide encoded by a nucleic acid molecule comprising apolynucleotide which hybridizes so the complement of the polynucleotideset forth in SEQ ID NO:1 wherein said hybridization occurs underconditions comprising hybridization in a buffer consisting essentiallyof 50% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA at 42° C. and wash in a solution consisting of 0.1×SSC at 65°C., and wherein said polypeptide stimulates the proliferation and/ordifferentiation of immune cells.
 47. The isolated polypeptide of claim46 comprising a heterologous polypeptide sequence.
 48. The isolatedpolypeptide of claim 47 wherein said heterologous polypeptide is that ofthe Fc domain of immunoglobulin.
 49. A composition comprising theisolated polypeptide of claim 46 and a pharmaceutically acceptablecarrier.
 50. An isolated polypeptide encoded by a nucleic acid moleculecomprising a polynucleotide which hybridizes to the cDNA in ATCC DepositNo. 209691 or 209641 wherein said hybridization occurs under conditionscomprising hybridization in a buffer consisting essentially of 50%formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA as 42° C., and wash in a solution consisting of 0.1×SSC at 65°C., and wherein said polypeptide stimulates the proliferation and/ordifferentiation of immune cells.
 51. The isolated polypeptide of claim50 comprising a heterologous polypeptide sequence.
 52. The isolatedpolypeptide of chum 51 wherein said heterologous polypeptide is the Fcdomain of immunoglobulin.
 53. A composition comprising the isolatedpolypeptide of claim 50 and a pharmaceutically acceptable carrier. 54.An isolated polypeptide comprising an amino acid sequence, wherein,except for one to 30 amino acid substitutions, said amino acid sequenceis identical to contiguous amino acid residues selected from the groupconsisting of: (a) amino acids residues +1 to +371 of SEQ ID NO:2; (b)amino acids residues +2 to +371 of SEQ ID NO:2; (c) amino acids residues+23 to +371 of SEQ ID NO:2; and (d) amino acids residues +23 to +231 ofSEQ ID NO:2; wherein the isolated polypeptide stimulates immune cellproliferation.
 55. An isolated polypeptide comprising an amino acidsequence, wherein, except for one to 30 amino acid substitutions, saidamino acid sequence is identical to contiguous amino acid residuesselected from the group consisting of: (a) an amino acid sequence of thefull length polypeptide encoded by the cDNA in ATCC Deposit No. 209691or 209641; (b) an amino acid sequence of the full length polypeptide,excluding the N-terminal methionine residue, encoded by the cDNA in ATCCDeposit No. 209691 or 209641; (c) an amino acid sequence of the maturepolypeptide encoded by the cDNA in ATCC Deposit No. 209691 or209641; andwherein the isolated polypeptide stimulates immune cell proliferation.56. An isolated polypeptide comprising a polypeptide consisting of afragment of SEQ ID NO:2 which fragment inhibits immune cellproliferation.
 57. An isolated polypeptide comprising a first amino acidsequence 90% or more identical to a second amino acid sequence of thesoluble extracellular domain of the polypeptide encoded by the cDNA inATCC Deposit No. 209691 or 209641, wherein the polypeptide comprisingsaid first amino acid sequence acts to inhibit immune cellproliferation.